THE ROLE OF THE BONE MARROW IN IMMUNOGLOBULIN A NEPHROPATHY

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1 THE ROLE OF THE BONE MARROW IN IMMUNOGLOBULIN A NEPHROPATHY Thesis submitted for the degree of Doctor of Medicine University of Leicester by Dr Katharine Buck BSc (Hons) MB BS MRCP Department of Medicine and Therapeutics University of Leicester May 2006

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3 Abstract The role of the bone marrow in IgA nephropathy Dr Katharine Buck IgA nephropathy (IgAN), the commonest glomerulonephritis in the western world, is characterised by mesangial deposition of IgA 1 molecules which probably arise in the bone marrow (BM). IgAl has a distinctive series of O-linked glycans, and abnormal patterns of this O-glycosylation are seen in serum and mesangial IgAl in IgAN. Lectin-binding and electrophoresis studies have indicated that the abnormality of IgAl O-glycosylation in IgAN takes the form o f reduced terminal galactosylation. O-galactosylation is effected by the intracellular enzyme (31,3galactosyltransferase (pl,3g T) during synthesis of IgAl by cells of the B lineage, and compromised activity of this enzyme may underlie altered O-glycosylation in IgAN. This study tested the hypothesis that undergalactosylation of IgAl is due to reduced B cell pl,3g T activity, focussing on the bone marrow, the putative source o f mesangial IgAl in IgAN. An assay for cellular pl,3g T activity, measuring the incorporation of ( 14C)galactose into asialo-ovine submaxillary mucin (aosm), was established and optimised. Peripheral blood (PB) and BM cell types were purified on antibody-conjugated magnetic beads, and their enzyme activity measured in comparison to a standard cell line. An initial set of experiments was performed using peripheral blood (PB) B and T cells from ten IgAN patients and ten controls with no difference in p 1,3GT activity being found. However, technical problems were encountered, which were addressed and improvements made. Subsequently, pi,3g T activity of BM B cells and reticulocytes, and PB B cells from 12 IgAN patients and 13 controls was measured. No abnormality of pl,3g T activity was seen in IgAN, despite demonstrating reduced terminal galactosylation of serum IgAl in the patient group. These results do not implicate abnormal function of pl,3g T as the cause of abnormal IgAl O-glycosylation in IgAN. Altered pi,3g T activity in a minor subset of cells, or abnormalities of other glycosytranferases, remain a possibility and should be investigated further.

4 To Simon, James, Georgie and Mairi

5 ACKNOWLEDGEMENTS I am indebted to John Feehally and Alice Smith for their supervision and help during this project; they were responsible for the original ideas, securing funding and overseeing the work. Both have also helped by reading the manuscript for which I am most grateful. Elaine Foster, Jonathan Barratt and Izabella Pawlucyk provided practical and emotional support and Alan Bevington helped me with cell culture and beta counting. I would like to thank Dr Thierry Hennet (Basel, Switzerland) and Dr Tony Corfield (Bristol, UK) for their kind help in establishing the assay to measure pl,3g T activity. Dr Corfield also generously donated desialylated and sialylated ovine submaxillary mucin and helped with troubleshooting the process o f its desialylation. I am particularly grateful to the patients who agreed to undergo bone marrow examinations, both those with IgA nephropathy and the controls; without their cooperation, none of the following work could have been performed. Mr Mike Allen, consultant in sports injuries and his anaesthetist, Dr David Raitt were always supportive and o f good humour. Finally, I am grateful to those people who had the confidence that I would complete this thesis and those who gave me the opportunity so to do (Drs Winney, Jenkins and Wood). I would like to acknowledge the patience of many friends and family members during the writing up period, especially my husband and my children.

6 The work was funded by the National Kidney Research Fund (Grant R47/1/97) and was performed between October 1997 and September This work has been presented at the American Society of Nephrology, San Diego, November 2003 and was published in abstract form: p i,3galactosy 1-transferase activity in IgA nephropathy. J Am Soc Nephrol 2003:14; 623A. Katharine S. Buck, Jonathan Barratt, John Feehally, Alice C. Smith.

7 INDEX Chapter One: Introduction Part I IgA Nephropathy and the bone marrow The clinical entity o f IgA nephropathy 1.1 Introduction Epidemiology Clinical features Natural history Histology Immunofluorescence studies Light microscopy Electron microscopy Treatment Immunosuppression Anti-hypertensive therapy Fish oil Transplantation 9 Pathogenesis of IgA nephropathy 1.7 The IgA immune system Mucosal IgA Serum IgA Differences between IgA 1 and IgA Human vs. rat IgA immune system Homing Marrow-mucosa axis Control o f IgA production 14 T cell subsets 15 Cytokines 15 CD40L IgA immune system in IgAN 16 V

8 1.8.1 Mesangial IgA Seram IgA Gastro-intestinal tract IgA production Bone marrow IgA production Tonsillar IgA production Immunisation studies Mechanisms o f IgAl deposition Immune mechanisms 21 Mesangial autoantigens 21 Macromolecular IgA Non-immune mechanisms 22 Physical properties of the IgA molecule 22 Direct interaction of IgA with mesangial matrix 22 Mesangial IgA receptor Mechanism o f glomerular injury Progression 24 Part II Glycosylation and the IgA l molecule 1.12 IgAl glycosylation Abnormalities o f IgAl in IgAN Lectin studies Mass spectrometry Analysis o f free glycans by size or charge separation Evidence suggestive that altered IgAl glycosylation is important in the 31 pathogenesis o f IgAN 1.15 Why should glycosylation be abnormal in IgAN? Glycosyltransferases and disease Rheumatoid arthritis The Tn polyagglutinability syndrome Malignancy pl,3g T activity in IgAN 35 Aim s o f the study 36 vi

9 Chapter Two: General Methods Cell culture and cell separation techniques 2.1 Density gradient centrifugation o f peripheral blood mononuclear cells Density gradient centrifugation o f bone marrow mononuclear cells Culture o f human PBMCs and BMMCs Culture o f THP-1 cells and U937 cells Separation o f cells with Dynabeads Cell preparation Dynabead preparation Bead-cell incubation Isolation and washing o f desired cell-type Cell lysis Standard cell lysate preparation Cytospin preparation and staining Staining with MGG Immunostaining with alkaline phosphatase Bone marrow smears 44 Protein chemistry and lectin binding 2.9 Protein assay Lectin binding o f serum IgAl Coating with prim ary antibody and blocking o f plates Samples Addition o f biotinylated lectins and avidin conjugated HRP Control to ensure constant level o f IgA l present Colour development and quantification 47 vii

10 Chapter Three: Development of p i,3 GT assay 3.1 Introduction Methodological considerations Materials and Methods Preparation o f the acceptor: asialo-ovine Submaxillary Mucin 52 Mild acid hydrolysis 52 Orcinol ferric chloride assay Evaluation o f the acceptor preparation Selection o f a standard cell lysate (3-1,3GT assay 55 Preparation of samples, standards and controls 55 Reaction mixture 55 Detection of f31,3gt activity 56 Calculation of (31,3GT activity in the test cell lysate samples Evaluation o f (31,3G T assay 57 Demonstration that cell lysates catalyse the incorporation of (14C)Gal 57 Necessity for a glycosidase inhibitor 5 7 Effect of halving reaction volumes 5 8 Reproducibility of the J31,3GT assay Results Evaluation o f acceptor Evaluation o f the acceptor preparation Evaluation o f the (31,3GT assay 62 Demonstration that cell lysates catalyse the incorporation of (u C)Gal 62 Necessity for a glycosidase inhibitor 65 Effect of halving reaction volumes 66 Reproducibility of the (31,3GT assay Conclusions Discussion 70 viii

11 Chapter Four: 31,3G T activity in peripheral blood lymphocytes of patients and controls 4.1 Introduction Subjects and Methods Subjects Samples and cell lysates controls Immunohistochemistry Determination o f optimal volume o f Dynabeads fl, 3 G T activity ofperipheral lymphocytes Reproducib il ity o f the fil,3g T assay Vicia villosa and Helix aspersa lectin binding o f serum o f IgA Statistical analysis Results Immunohistochemistry Determination o f optimal volume o f Dynabeads p l,3 G T activity o f peripheral lymphocytes Reproducibility o f the p i,3 G T assay Relationship between the fl,3 G T activity o f test cell- and THP-1 86 lysates HA and VV lectin binding o f serum o f IgA I Conclusions Discussion 88 ix

12 Chapter Five: (31,3 GT activity in bone marrow and blood of patients and controls 5.1 Introduction Subjects and M ethods Subjects Samples and controls 95 Bone marrow 95 Peripheral blood Immunohistochemistry Determination o f optimal volume o f Dynabeads p i,3g T activity o f bone marrow B cell precursors and controls Reproducibility o f p i,3 G T activity Lectin binding o f serum o f IgA Statistical analysis Results Immunohistochemistry Determination o f optimal volume o f Dynabeads (31,3GT activity o f bone marrow B cell precursors Reproducibility o f p i,3 G T activity Relationship between the p i,3 G T activity o f test cell- and THP lysates Lectin b inding o f serum o f IgA Correlation between p l,3 G T activity and lectin binding Conclusions Discussion 111 X

13 Chapter Six: Discussion Summary o f findings Interpretation o f findings There is an undetected difference in activity o f J31,3GT activity There is no difference in j3l,3g T activity Conclusion 121 Appendix One Buffers and solutions 123 Appendix Two Bone marrow protocol 128 References 129 xi

14 INDEX OF FIGURES Schematic representation o f the IgAl monomer, indicating possible sites of O-glycosylation on the hinge region The structure of the human IgAl hinge region Orcinol-ferric chloride assay: typical standard curve pl,3g T assay to compare the functioning o f home-desialylated OSM with the standard donated by Dr Corfield pi,3g T activity o f U937 lysate: typical standard curve The effect o f using the glycosidase inhibitor dimercapto propan-l-ol (DMP) on pl,3g T activity The effect o f halving the reaction volumes on p 1,3GT activity B cells separated from peripheral blood by density gradient centrifugation and antibody-conjugated magnetic beads T cells before and after separation Monocytes before and after overnight incubation The relationship between bone marrow B cell pl,3g T activity in IgAN patients and controls and HA lectin binding INDEX OF TABLES Summary of the reported abnormalities of lectin binding in IgAN The reduction in sialic acid content of sialyl-osm (sosm) as it underwent progressive desialylation Construction of standard curve for pl,3g T assay using U937 cell lysate Intra-assay and inter-assay coefficients of variation (CV) of pl,3g T 68 assay of THP-1 activity in AU/pg Final composition o f substrate solution 70 xii

15 The percentage o f cell types by immunohistochemistry at various 79 stages o f cell separation prior to measuring pl,3g T activity B cell counts achieved using different volumes o f antibodyconjugated magnetic beads for subjects A and B Maximum B cell protein yield using a range o f volumes o f CD 19- magnetic beads pi,3g T activity of patient and control peripheral blood B and T cell lysates (AU/pg) and o f THP-1 cell lysates The relationships between test cell pl,3g T activity and that of THP-1 86 lysates HA and VV lectin binding of patient and control serum IgAl expressed as OD at 492nm Individual details of patients and controls undergoing bone marrow aspirate The percentage of bone marrow cells staining positive by immunohistochemistry at three different stages of separation Maximum B cell protein yield using a range of volumes o f CD 19 magnetic beads pi,3g T activity of patient and control bone marrow-b cell, peripheral blood-b cell and reticulocyte lysates in AU/pg Table showing the dates and sequence of experiments and the CV of 105 THP-1 pl,3g T activity over the three month series of experiments The relationships between test cell pl,3g T activitiy and that of 106 THP-1 lysates Lectin binding of patient and control IgA (expressed in O D ) The relationships between test cell pl,3g T activities and HA and W lectin binding xiii

16 ABBREVIATIONS pi,3glcnac pl,3g T P 1,3 N-acetylglucosaminyl p 1,3galactosyltransferase AC Amaranthus caudatus ACE angiotensin converting enzyme aosm asialo-ovine submaxillary mucin ASGPR asialoglycoprotein receptor ATCC American Type Culture Collection AU arbitrary units BM bone marrow BMMC bone marrow mononuclear cells BSA bovine serum albumin C l inh C l inhibitor C3 complement component 3 CIC circulating immune complexes CMV cytomegalovirus Cosmc core 1 GT specific molecular chaperone CV coefficients o f variation DMP dimercaptol propan-l-ol DPM disintegrations per minute ECACC European Collection o f Cell Cultures ECL Erythrinia cristagalli lectin ELISA enzyme-linked sandwich immune assay ESRF end stage renal failure FCS fetal calf serum Gal galactose GalNAc N-acetyl galactosamine GalNAcase N-acetylgalactosaminidase GalNAcT N-acetylgalactosamine-transferase GALT gut-associated-lymphoid-tissue GlcNAc N-acetylglucosamine GN glomerulonephritis

17 GPR GT HA HBSS HC1 HEPES HIV HLA Hp HPLC HSP IgA IgAN IgG IgM ISH KOH MALDI-ToF-MS MBP MC MGG MHC mm M nch MRC N NaOH NeuNAc NSAIDs O OD PBMC general purpose reagent galactosyltransferase Helix aspersa Hank s balanced salt solution hydrochloric acid 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid human immunodeficiency virus human leucocyte antigen Helicobacter pylori high performance liquid chromatography Henoch-Schdnlein purpura immunoglobulin A immunoglobulin A nephropathy immunoglobulin G immunoglobulin M in situ hybridisation potassium hydroxide matrix-assisted laser desorption ionisation time-of-flight mass spectrometry mannan binding protein mesangial cell May-Grunwald/Giemsa major histocompatibility complex milimolar manganese chloride Medical Research Council nitrogen sodium hydroxide sialic acid non-steroidal anti-inflammatory drugs oxygen optical densitiy peripheral blood mononuclear cells XV

18 PBS plga PMFP PNA PP PTA RA RCT rpm RT SC SD SDS-PAGE sem Ser SFC SLE sosm SRBC TBS TCA IF TGFp Thr UDP W phosphate buffered saline polymeric IgA permanent mixed field polyagglutinability peanut agglutinin Peyer s patches phosphotungstic acid rheumatoid arthritis randomised controlled trial revolutions per minute room temperature secretory component standard deviation SDS-polyacrylamide gel electrophoresis standard error of the mean serine spot forming colonies systemic lupus erythematosus sialyl-osm sheep red blood cells Tris buffered saline trichloroacetic acid Thomsen-Friedenreich transforming growth factor p threonine uridine diphosphate Vicia villosa xvi

19 Chapter One Part 1 IgA Nephropathy and the bone marrow The clinical entity of IgA nephropathy 1.1 Introduction In 1965, when the use of fluorescein-labelled antibodies was in its infancy, Bodian and colleagues first described diffuse deposits of immunoglobulins in the glomerular mesangium occurring in association with focal and segmental glomerulonephritis in children with recurrent macroscopic haematuria (Bodian et aly 1965). The following year Berger presented data on mesangial deposits of immunoglobulin A (IgA) at the 1966 International Congress in Washington (Berger et al, 1967) and in 1968 his classic description of IgA nephropathy (IgAN) was published (Berger & Hinglais, 1968), known initially as Berger s disease. IgAN is now recognised as the commonest glomerulonephritis (GN), in developed countries (d Amico, 1987), accounting for a significant proportion of the world s end stage renal failure population. In the subsequent 35 years much has been learnt about the natural history of the disease and the function of the IgA immune system in health and in IgAN, however fundamental questions about the pathogenesis of this disease remain unanswered. Diagnosis depends on the demonstration of dominant or co-dominant deposition o f IgA in the glomerular mesangium in a renal biopsy. Mesangial IgA deposits may also be found in association with Henoch-Schonlein purpura (HSP), a systemic vasculitis and also systemic lupus erythematosus (SLE) and alcohol related liver disease. To establish the diagnosis of 1

20 primary IgAN the latter conditions must be excluded. Increasingly HSP is regarded as part o f the spectrum o f IgAN. IgAN is also found in association with a variety of other conditions such as ankylosing spondylitis, coeliac disease and dermatitis herpetiformis (van Es, 1997). The significance of these associations is not fully understood. This thesis will restrict itself to primary IgAN. 1.2 Epidemiology IgAN is found worldwide and accounts for 15-50% of all GN. The incidence varies considerably, although much o f this variation can be explained by differing access to health care and varying biopsy policies. Generally the incidence is thought to be highest in the Pacific-rim countries, such as Singapore and Japan, which both have well established urine-screening programs. 2% of army recruits in Singapore were found to have asymptomatic urine abnormalities. All underwent renal biopsy. IgAN was diagnosed in 50% (Kincaid-Smith & Nicholls, 1983). In Britain IgAN accounts for about 20% of primary GN (Power et al, 1985; Propper et al, 1987). Johnston et al (1992) used the Medical Research Council s (MRC) Glomerulonephritis Registry to examine the picture nationally and reported that IgAN was found in 9% of all biopsies, which was 18% of primary GN, thus making the British experience broadly similar to that in Europe, where a large Italian series reported that IgAN represented 23% of all patients with primary GN (d Amico et al, 1985). Similarly, in New Zealand, 16% of patients reported in the New Zealand Collaborative GN Study had IgAN (Bailey et al, 1994). The presenting feature in these large series is most commonly microscopic haematuria (around 60% in Britain and New Zealand), it is therefore easy to understand that the apparent incidence o f the disease 2

21 will depend on referral and biopsy practices for subjects with asymptomatic urinary abnormalities. Men are more frequently affected than women, 70% of the patient group reported by d Amico were male (d Amico et al, 1985), similar to that found in the UK (77%) (Johnston et al, 1992). This ratio is likely to be explained in part by screening practices. There are also racial variations in incidence; Polynesians in New Zealand have a lower incidence of IgAN than Caucasians, despite having a higher overall incidence o f all types of GN (Bailey et al, 1994). The incidence appears to be lower in Blacks, both North American and African and prevalence has been reported to be low in Australian Aborigines (Galla, 1995). IgAN has been associated with several phenotypes of the HLA MHC, although these associations are not consistent around the world. HLA-B12 has been reported in association with IgAN in the United States and HLA-B35 has been associated with a poor prognosis in France (Berthoux et al, 1993). Other associations exist for other parts of the world. In addition the ACE genotype DD may be associated with a more rapid decline in renal function (Hunley et al, 1996). There have been a number of reports that IgAN may have a genetic component. Family clusters have been described, one of the largest of which was in Kentucky (Julian et al, 1985). Three potentially related pedigrees were described, which contained 14 patients and a further 17 patients with clinical GN. The authors were able to exclude common environmental factors as an explanation. Interestingly there was a lack of a common HLA genotype. There are also reports of significant numbers of relatives of patients with IgAN 3

22 having persistent microscopic haematuria (15.6%) and proteinuria (3%) or both (4%) (Schena, 1993). However, despite these reports 90% o f cases are sporadic (Ibels & Gyory, 1994). 1.3 Clinical features IgAN typically presents in young men either with an asymptomatic finding of microscopic haematuria and proteinuria or episodic macroscopic haematuria. In a large series mean age at presentation was 39 years (Ibels & Gyory, 1994). The male to female ratio was 1.81 with the range quoted by others lying between 1.1 and 10.3 (van Es, 1997). Both age and sex incidence may again depend on screening practices. 40% of patients presented with macroscopic haematuria, which is characteristically preceded by infections of the respiratory tract or other systems. Loin pain is experienced by about a third of patients. Microscopic haematuria is found in 87% of patients at presentation. Low-grade proteinuria (1 to 3g per day) is common, being found in 47% of patients (van Es, 1997). Nephrotic range proteinuria is rarer and occurred in 16% (Ibels & Gyory, 1994). Impaired renal function at presentation is relatively common, found in 36%; hypertension was found in 31% of patients at the time of diagnosis (Ibels & Gyory, 1994). 1.4 Natural history Although initially IgAN was considered a benign disease, there is now widespread agreement in the literature that the course of IgAN tends to be of slowly progressive deterioration in renal function usually associated with hypertension. Patients affected by IgAN are estimated to account for 10% o f the world's end stage renal failure (ESRF) 4

23 population (d Amico, 1987). About 25% of patients require maintenance dialysis 20 years after the apparent onset of the disease. Others have suggested a better prognosis: In New Zealand Bailey et al, (1994) found an actuarial renal survival rate of 91% at 10 and 15 years and concluded that patients with a poor prognosis can be identified with considerable certainty at presentation. There is agreement on some features carrying a poor prognosis, namely, raised creatinine at presentation, hypertension and proteinuria greater than one gram (Johnston et al, 1992; d Amico, 1992; Ibels & Gyory, 1994). Other features remain more controversial; some authors find that male sex (d Amico, 1992) and older age at diagnosis (Johnston et al, 1992; Ibels & Gyory, 1994) have a worse outcome. Most agree that episodic macroscopic haematuria carries a favourable prognosis with the exception of Bennett and Kincaid-Smith (1983) who found it associated with a significantly worse outcome, which can perhaps be explained by the fact that 1 0 0% of patients biopsied at the time o f haematuria were found to have crescents. 1.5 Histology The diagnosis o f IgAN can only be made with certainty by examining a renal biopsy specimen Immunofluorescence studies It is the demonstration of dominant or co-dominant IgA deposits in the glomerular mesangium on immunofluorescence, which is the hallmark of the disease. C3 and other terminal complement components are nearly universally present and IgG and IgM are commonly found in the same distribution as IgA. C lq and C4 are absent or only weakly present, indicating inactivity o f the classical pathway o f complement. On light microscopy 5

24 the most characteristic and frequent abnormality is mesangial enlargement, produced by a combination of excess matrix and hypercellularity (d Amico, 1987). Most commonly mesangial proliferation is focal and segmental although it may be diffuse Light microscopy Biopsy samples taken at an early stage of the disease with only mild mesangial abnormalities have few polymorphonuclear and mononuclear cells in the glomeruli. The mononuclear cell count increases as the glomerular lesions become more severe and there is a correlation between the number o f macrophages and the presence o f crescents and proteinuria. As the disease progresses macrophages and T cells are found in the tubulointerstitium and these cells have therefore been implicated in mediating scarring. In more advanced stages of the disease tubular atrophy and interstitial fibrosis are found leading to widening of the interstitial space. The width of the cortical interstitium correlates with the serum creatinine (van Es, 1997). Crescents may be found if the biopsy is taken at the time o f macroscopic haematuria or impaired renal function (d Amico, 1987) Electron microscopy Electron dense deposits are found in the mesangial and paramesangial areas on electron microscopy and in the capillary walls. Changes of the glomerular basement membrane such as thinning and splitting or thickening are also commonly seen (d Amico, 1987). Fusion o f the epithelial cell foot processes may be present. Renal biopsy features bestowing a bad prognosis include increasing numbers of sclerosed glomeruli or with segmental sclerosis and adhesions (Ibels & Gyory, 1994). The MRC GN registry found that patients with focal and segmental proliferative changes had a reduced 6

25 chance of renal survival compared to those with diffuse mesangial proliferation (Johnston et al, 1992). Tubular atrophy, interstitial fibrosis and inflammation and blood vessel thickening have all been found to be associated with an adverse outcome (Ibels & Gyory, 1994). Despite these correlations no single biopsy feature has been found to be predictive of clinical outcome. D Amico found an overlap of biopsy findings in two groups of patients, one of which progressed, while the other group maintained normal serum creatinine (d Amico, 1985). Yoshikawa et al, (1990) in a repeat biopsy study looking at a group of children whose urinary abnormalities regressed in comparison to a group with continuing haematuria and proteinuria found that the initial biopsy findings were essentially the same in the two groups. 1.6 Treatment As understanding of pathogenesis improves there is greater interest in potential diseasespecific treatments, however none is yet available. When considering treatment the fact that IgAN is a disease with a variable outcome, which is slowly progressive and affects a predominantly young population must be borne in mind. It is therefore likely to be necessary to treat a relatively healthy group o f patients for a prolonged period to affect the natural history. Therefore any treatment must have a low risk of toxicity. Many of the features of a poor prognosis are in effect markers of existing damage, namely, a raised serum creatinine, heavy proteinuria, hypertension and glomerular and interstitial sclerosis on biopsy; if treatment were directed at this group it might be too late to modify the disease process. 7

26 1.6.1 Immunosuppression With the rationale o f suppressing the IgA immune system, there has been considerable interest in the use of conventional immunosuppressive agents. Recently interest has focused on the prolonged use (between four and 24 months) of moderate doses (0.25- lg/kg/day) of corticosteroids sometimes in combination with cytotoxic agents (reviewed Floege, 2003i). The literature now supports the use of steroids in certain patient groups, although there are still only two randomised controlled trials (RCT) providing evidence that clinical outcome is altered. These trials have not always documented other factors determining progression, namely, hypertension (the most important), lipids, the avoidance of nephrotoxic drugs especially non-steroidal anti-inflammatory drugs (NSAIDs) and smoking cessation (Floege, 2003i). Aggressive supportive treatment is probably of greater benefit than immunosuppression and is associated with less toxicity. Immunosuppressive regimens probably have a better risk: benefit ratio in the case of rapidly progressive renal failure associated with crescentic IgA nephritis although there is no controlled data to prove this (Feehally, 1999) Anti-hypertensive therapy The recommendation today is a target blood pressure (BP) of 125/75 mmhg in patients with renal disease and greater than lg proteinuria/day and there is non-rct evidence to support this goal (Floege, 2003i). Evidence is also emerging to support the prejudice that renin-angiotensin blockade has a beneficial effect on renal function independent of blood pressure (Praga et al, 2003; Park et al, 2003). Angiotensin-converting enzyme (ACE) inhibitors are therefore now the agents of first choice in the treatment of hypertensive IgAN patients. Additional benefit has been demonstrated by the addition of Angiotensin II receptor blockers (Campbell et al, 2003; Nakao et al, 2003).

27 1.6.3 Fish oil Many in vitro effects of omega 3 fatty acids have been described which should be of benefit in reducing progression of chronic renal failure (Feehally, 1999). One large study showed benefit of treatment (Donadio et al, 1994), but a further trial funded by the National Institutes of Health, which examined the effect of fish oil or prednisolone versus placebo showed no benefit of fish oil (or prednisolone) with regard to creatinine clearance although proteinuria (assessed by urine protein.creatinine ratio) fell in both treatment groups (Hogg et al, 2003) Transplantation End stage renal failure patients with IgAN undergo transplantation and anecdotally the disease was said to recur only rarely, which was attributed to the effects of the immunosuppressive drugs used. However, there is now evidence that histological recurrence occurs in up to 60% o f patients which, in a review of several studies including almost 1200 patients with underlying IgAN, manifested as graft dysfunction in approximately 13% and lead to graft loss in about 5% (Floege, 2004). The major risk factor for recurrence-related graft loss is time from transplantation and no specific intervention has been shown to be of benefit in preventing histological recurrence. Controversy exists as to whether living-related donor kidneys are at higher risk of disease recurrence than kidneys from non-related donors. Nonetheless, the survival of either type o f graft is excellent in patients with IgAN (Floege, 2003ii). 9

28 Pathogenesis of IgA nephropathy The fundamental questions in the study o f IgAN are: What causes IgA to deposit in the glomerular mesangium? Why does deposited IgA cause mesangial proliferation, expansion, glomerulosclerosis, interstitial fibrosis and renal fa ilu re? IgAN is thought to be primarily an abnormality of the immune system and not of the kidney: numerous abnormalities of immune function have been described, the disease frequently recurs in renal transplants, first described by Berger in 1975 (Berger et al, 1975; Odum et al, 1994) and when kidneys containing IgA deposits were transplanted inadvertently into non IgAN patients, the IgA deposits had significantly reduced when the histology was re-examined (Sanfilippo et al, 1982; Silva et al, 1982). For these reasons the following two sections will examine the IgA immune system in health and IgAN. 1.7 The IgA immune system IgA is found in two different compartments: in the mucosae it is the predominant antibody of secretions, providing defence against micro-organisms and toxins. IgA is also found in the blood, the function o f which remains poorly understood. IgA exists in two isotypic forms, IgAl and IgA2, with IgA2 occurring as two allotypic variants, IgA2m(l) and IgA2m(2). Each o f these forms is found in various degrees o f polymerization (Kerr, 1990). 10

29 1.7.1 Mucosal IgA The majority of IgA is produced at the mucosal surfaces, of which the quantitatively most important site is the gastrointestinal tract (Conley & Delacroix, 1987). Mucosal IgA is predominantly (95%) polymeric (plga), in which monomers are connected by a 16 KD J chain to form dimers. In addition, secretory component (SC), which is produced by epithelial cells, is covalently bound to the J-chain-associated dimer. SC is part of a cell surface polymeric Ig receptor, which mediates secretion of IgA into the lumen by transcytosis across the epithelial cell barrier. SC has been shown to markedly stabilise the structure of IgA and increase its resistance to proteolysis (Kerr, 1990). The IgA subclass distribution varies with different mucosal locations, from 67% IgAl in the bronchial secretions to only 35% IgAl in colonic fluid (Kerr, 1990). In secretions IgA prevents microorganisms and foreign proteins from adhering to and penetrating the mucosal surfaces. It also neutralises toxins and infectious organisms (Conley & Delacroix, 1987) Serum IgA The major source o f human serum IgA is the bone marrow. More IgA than IgG is secreted by in vitro cultures of unstimulated marrow (Conley & Delacroix, 1987). Peripheral lymph nodes, including the tonsils and the spleen also contribute to serum IgA production. It is predominantly monomeric with an IgAl :IgA2 ratio of about 9:1 (Kerr, 1990). The role of serum IgA remains uncertain. The majority of subjects deficient in IgA are asymptomatic, although there is an increased incidence of allergy and autoimmune diseases. Serum IgA may remove the small amounts of antigen that continuously leak into the circulation before the antigen is able to elicit an IgM or IgG response and thus it supplements the process of oral tolerance. Others however have suggested completely different roles for serum IgA such as the enhancement o f the immune response (Conley & Delacroix, 1987). 11

30 1.7.3 Differences between IgA 1 and IgA2 There are a number of structural differences between IgAl and IgA2, which differ in only 22 amino acids in man. This is mainly due to the absence of the 18 amino acid hinge region in IgA2, which in the IgAl molecule lies between the CHI and CH2 regions of the heavy chain (Kerr, 1990). The IgAl hinge region is now thought to be an insertion into the phylogenetically older IgA2 (Tomana et al, 1999). It is composed of an unusual repeating sequence rich in proline, serine and threonine. Serine and threonine are capable of forming O-linked bonds with oligosaccharides, of which the IgAl molecule has up to ten per hinge region, shown in Figure 1.1. Both IgAl and IgA2 carry N-linked sugars, IgAl has 2 per a- chain, while IgA2m(l) has 4 and IgA2m(2) 5 per a-chain (Kerr, 1990). Fab Hinge region Fc C H1 Ch2 Ch3 Light chains Tailpiece CH1 Pro Ser ^O -glycan Thr ^-O-givcan Pro Pro Thr ^-O-glycan Pro Ser «-0-glycan Pro Ser «ro-glycan Thr ^O-glycan Pro Pro Thr ^O-glycan Pro Ser «-0-glycan Pro Ser 4-0-glycan Ch2 Figure 1.1 Schematic representation of the IgAl monomer, indicating possible sites of O-glycosylation on the hinge region (from Allen, 1998). 12

31 1.7.4 Human vs. rat IgA immune system There are fundamental differences in the structure and metabolism o f IgA between animals and man. For example no non-primate species has a hinge region analogous to human IgAl. Unlike in humans, most of the plga in the serum of rats appears to be derived from plasma cells in the lamina propria of the gut. It enters the serum via the mesenteric lymph and the thoracic duct (Conley & Delacroix, 1987). Rat serum IgA is at a reduced concentration in comparison to man and appears to be equally divided between the monomeric and polymeric form. Clearance mechanisms of serum IgA are also fundamentally different in the rat; there is an active mechanism for the secretion of circulating plga into the bile, which is therefore highly enriched for plga. In man only a tiny proportion of serum IgA is secreted into the bile, the majority being cleared from the vascular compartment (Conley & Delacroix, 1987). Such work shows that data from animals about IgA metabolism and kinetics cannot be readily extrapolated to humans Homing Initially it was believed that the mucosal and systemic IgA compartments were separate but more recently it has been understood that the two are in complex relationship to one another. Lymphocytes o f the gastrointestinal tract are first exposed to antigen at the mucosal surface in Peyer s patches (PP), part of the gut-associated-lymphoid-tissue (GALT). Antigens are taken up by the specialised epithelial cells (M cells), which overlie the lymphoid nodules of PP, where the B cells are primed to become secretory IgA-B cells. They enter the circulation, both the peripheral circulation and the lymphatics (Pabst & Binns, 1981) and then home to diverse mucosal effector sites such as the lamina propria, where they mature into plasma cells and secrete IgA (Brandtzaeg et al, 1998). The control o f homing is incompletely understood but is related to homing receptors and integrins. This 13

32 process of lymphocyte recirculation is thought to be an important part of the immune response as it distributes antigen-reactive, unprimed and memory cells throughout the body such that contact with local antigens is facilitated and memory cells are ubiquitous Marrow-mucosa axis There is growing awareness that homing of B cells is not only to the mucosal surfaces, but also to systemic sites such as the bone marrow. Mice that underwent chronic oral stimulation with sheep red blood cells (SRBC) had a significant IgA plaque-forming cell response in the bone marrow. This was not the case in mice which underwent various systemic immunisations nor those which received only 2 days of oral SRBCs followed by an intra-peritoneal injection (Alley et al, 1986). Pabst and Binns (1981), in an in vivo lymphocyte labelling experiment in pigs, also showed a significant and surprisingly high number of fluorescein-labelled lymphocytes in the bone marrow after injection of mesenteric lymph nodes. This suggested that significant numbers of bone marrow lymphocytes are members of the recirculating pool and that there is communication between the two parts o f the IgA immune system. Evidence of a similar axis in man comes from a nasal immunisation study, which demonstrated the presence of specific IgAl antibody-producing cells in the bone marrow after nasal antigen presentation (de Fijter et al, 1996) Control o f IgA production In mice a large number of studies have strongly suggested that various populations of T cells and their cytokines influence the initial commitment and differentiation of B cells to IgA plasma cells (McGhee et al, 1989). Much of the data on the control of mucosal IgA 14

33 production and synthesis is derived from the study of knockout mice, thus a degree of caution is necessary in extrapolating the findings to man. T cell subsets CD4+a(3 T cells are essential for induction and regulation of the IgA mucosal response. TCRp knockout mice contain almost no mucosal IgA producing cells (Fujihashi et al, 1996). A significant reduction in IgA synthesis was noted in mice depleted of their CD4+ T cells (Mega et al, 1991). y5 T cells also play an important role in the control of IgA production. TCR 8 7 knockout mice exhibit reduced numbers of IgA plasma cells and display markedly reduced IgA responses to oral immunisation, despite normal a p T cells (Fujihashi et al, 1996). Cytokines The Th2 subset of CD4+ T cells, which preferentially produce IL-4, IL-5, IL-6 and IL-10 promote IgA responses (McGhee et al, 1989; Lycke 1998). That these cytokines are produced by multiple cells hampers our understanding of cellular interactions in the mucosa (Lycke, 1998). TGFp is also important in IgA production, leading to B cell surface-iga (slga) expression and subsequent IgA secretion. Experimentally, the effect of TGF-P on secretion depends on the method of B cell stimulation, although it produced a consistent effect in increasing siga+ B cells (Ehrhardt et al, 1992). CD40L CD40-CD40L interaction is also important in controlling B cell differentiation (Lycke, 1998). It appears that while the IgA isotype switch may proceed in the presence of cytokines alone, terminal differentiation may be critically dependent on cell membrane 15

34 CD40 interaction with activated CD40L-expressing T cells (Lycke, 1998). Thus a close interaction with the activated T cell is probably a necessary precondition for IgA B cell differentiation. 1.8 IgA immune system in IgAN Mesangial IgA Mesangial IgA is predominantly polymeric IgAl. The polymeric nature of the deposits was established by the demonstration of J chain by immunohistochemical techniques (Komatsu et al, 1983; Tomino et al, 1982i; Bene et al, 1982; Lomax-Smith et al, 1983), although poor specifity of some anti-j chain antibodies and the co-deposition of IgM (polymeric IgM is also connected by J chain) caused confusion. Others have used the binding of free secretory component (SC) (Bene et al, 1982; Egido et al, 1980) or elution studies (Tomino et al, I982i; Monteiro et al, 1985) to demonstrate the polymeric nature of mesangial IgA. Immunohistochemistry was also used in the demonstration of the IgA subclass, again with some conflicting results attributed to poor antibody specificity (Lomax-Smith et al, 1983; Conley et al, 1980; Tomino et al, 1981). The later studies, using monoclonal antibodies agreed that IgAl was the predominant subclass deposited (Valentijn et al, 1984; Russell et al, 1986; Rajaraman et al, 1986). Because plga has restricted origins within the IgA immune system, its presence in the kidney may provide information about the site and nature of the immune abnormalities that underlie IgAN and has led to the study of IgA-producing sites in IgAN. In keeping with the idea that IgAN is primarily an abnormality o f the immune system are the multiple 16

35 abnormalities of the IgA immune system. These are best described by examining the different compartments in which IgA is found Serum IgA Total serum IgA levels are increased in % of cases (d Amico, 1988). The increase is restricted to the IgAl subclass (van den Wall Bake et al, 1988ii, Layward et al, 1993) with a predominance of X light chains (Lam et al, 1991). The majority of serum IgA is monomer (Harper & Feehally, 1993) although there is an increase in both plga and mlga (van den Wall Bake et al, 1989i). Most studies report an increase in macromolecular ( kda) forms of serum IgA in most patients with IgAN. During exacerbations of disease activity there may be further increases in the levels o f IgA macromolecules despite there being no demonstrable change in total IgA. It is uncertain what proportion of the macromolecular IgA is uncomplexed plga, IgA-immune complexes, IgA-rheumatoid factor or rarely, IgA cryoglobulins (Feehally, 1988) Gastro-intestinal tract IgA production Because the mucosa is the main site of plga production and because of the occurrence of macroscopic haematuria at the time o f mucosal infections (d Amico, 1987) initially it was believed that the IgA deposited in the kidney in IgAN derived from the mucosal surfaces. This was supported by the increased levels of circulating IgA antibodies to food and common mucosal antigens (Galla, 1995). However studies published in the late 1980s and early 1990s revealed reduced IgA plasma cells in duodenum of IgAN patients. Harper developed an in situ hybridisation (ISH) technique to look for J chain mrna (indicating polymeric immunoglobulin synthesis) in IgA plasma cells, which he applied to the duodenum o f patients with IgAN. (Harper et al, 1994i). Combined ISH and 17

36 immunofluorescence revealed a reduction in J chain mrna-positive IgA plasma cells, which argued against the gastrointestinal mucosa being the source of glomerular IgA. These results corroborated those of a previous study, which also found a reduced proportion of IgA cells in the lamina propria in IgAN (Hene et al, 1988) Bone marrow IgA production The bone marrow (BM) is the major source of human serum IgA in health (Kerr, 1990) and is now thought to be the likely origin of mesangial IgA in IgAN. Immmunofluorescence studies have revealed that patients have an increased proportion of IgAl plasma cells in the bone marrow (van den Wall Bake et al, 1988i). A two-colour immunofluorescence study on bone marrow trephine biopsies demonstrated an increased percentage of IgA plasma cells in IgAN patient biopsy specimens compared to controls. IgAl plasma cells were disproportionately increased in patients (Harper et al, 1994ii). There was a positive correlation between the percentage of marrow IgA plasma cells and serum IgA levels in patients. The results are suggestive that the raised circulating levels of IgA originate from the bone marrow. A further combined immunofluorescence in situ hybridisation study revealed a greater proportion of J chain mrna positive IgA plasma cells in the bone marrow o f IgAN patients, although the absolute numbers of J chain expressing cells were unchanged (Harper et al, 1996). These data suggest that the excess production of dimeric IgA may originate in the BM in IgAN, which is further supported by the finding of significantly higher numbers of total IgA and IgAl spot forming colonies (SFC) in bone marrow aspirates of IgAN patients (de Fijter et al, 1996). There were excellent correlations between the serum levels of IgA and IgAl and the numbers of the respective SFCs. 18

37 1.8.5 Tonsillar IgA production The tonsils have been a focus of study in IgAN, in which a marked increase in tonsillar IgA- and a decrease in IgG-bearing lymphocytes have been described (Egido et al, 1984). There was a significant increase in tonsillar cells simultaneously binding IgA and secretory component i.e. producing plga. Two studies by Bene and colleagues corroborated the altered proportions of IgA- and IgG-secreting cells (Bene et al, 1983 and 1991i). Harper et al, also found a greater proportion of J chain mrna-positive interfollicular IgA cells in the tonsils of IgAN patients (Harper et al, 1995). There is now limited evidence from Japan that tonsillectomy may improve renal survival rate (Xie et al, 2003) Immunisation studies As outlined above the IgA immune system is traditionally subdivided into systemic and mucosal compartments, but from the description o f priming of B cells, migration through the systemic circulation and homing back to the mucosal effector sites, it is clearly a dynamic system with considerable overlap between the compartments. Immunisation studies provide in vivo information about this dynamic system. Antigen challenges to the systemic IgA immune system by subcutaneous or intramuscular immunisation result in an exaggerated IgAl response in IgAN (Barratt et al, 1999). However systemic IgAl responses after limited mucosal antigen challenge are deficient (de Fijter et al, 1996; Roodnat et al, 1999). It has previously been noted that pre-immunisation titres of antibodies to recall antigens are higher in IgAN patients (van den Wall Bake et al, 1989ii) so it would therefore appear that patients have a greater memory pool of IgAl-producing B cells to certain parenteral recall antigens. The hypothesis of mucosal hyporesponsiveness with reactive or compensatory systemic hyperresponsiveness after prolonged or repeated stimulation is further supported by data on the systemic response to chronic Helicobacter 19

38 pylori (Hp) infection in IgAN (Barratt et al, 1999). Patients infected with Hp had a significantly higher IgA seropositivity rate than healthy controls and mean serum anti-hp IgA antibody levels were significantly higher in patients. The response was predominantly IgAl and was polymeric in both patients and controls. In conclusion, there is clear evidence of disordered IgAl production in IgAN with decreased gastro-intestinal mucosal production and increased production in systemic immune sites such as the bone marrow and the tonsils. Serum levels of IgAl to food antigens and a chronic mucosal infection (Hp) are elevated, but the response to a one-off antigen such as cholera toxin subunit B or Salmonella are diminished. It is now believed that the IgAl deposited in the glomerular mesangium in IgAN derives from the bone marrow, probably because of a disordered marrow-mucosa axis, which leads to ectopic production of "mucosal" type antibody. Further work is currently being undertaken on homing of lymphocytes in IgAN. What causes IgA to deposit in the glomerular mesangium? 1.9 Mechanisms of IgA l deposition IgA deposition does not occur simply because of the elevated levels of plga; IgA levels may be high in both IgA myeloma and HIV disease, but clinical IgAN is rare in these conditions (Bene et al, 1991 ii). Traditionally mechanisms of deposition have been considered as either immune or non-immune; more recent evidence favours the latter mechanisms.

39 1.9.1 Immune mechanisms Mesangial autoantigens Antibodies to several viruses including herpes simplex, hepatitis B and CMV and to several foods including soy, cows milk and rice proteins have been detected in the mesangium of IgAN patients. Nonetheless, the data show that no specific microbial or food antigen has been identified in the glomeruli of IgAN patients (Galla, 1995). Neither is there convincing evidence of circulating IgA autoantibodies against a mesangial antigen (O Donoghue & Feehally, 1995). Thus, serum and mesangial antibodies are polyclonal and not indicative o f specific antibody deposition. Macromolecular IgA There is however increasing recognition that IgAl-self-aggregates and immune complexes are of pathogenic importance in mesangial deposition (Tomana et al, 1999; Kokubo et al, 1998) but are probably deposited due to non-immune mechanisms. Such complexes are too large to pass through endothelial fenestrae to enter the space of Disse to be cleared via the hepatocyte asialoglycoprotein receptor (ASGPR) (Tomana et al, 1999). Glomerular deposition may be by non-specific size-dependant mesangial trapping, however a recent study showed that mesangial cells bind high molecular weight IgAl with high affinity (Leung et al, 2002ii). Thus macromolecule formation may favour deposition and is associated with abnormalities o f glycosylation o f the IgAl molecule (see Section 1.14). 21

40 1.9.2 Non-immune mechanisms Physical properties of the IgA molecule The lack of evidence that mesangial IgA deposition is because of conventional immune mechanisms led researchers to consider other mechanisms to explain the phenomenon, such as the physical properties of the IgAl molecule. Electrical charge IgA eluted from renal biopsies is anionic with a restricted pi (4.5 to 5.6), which contrasts with the broader and more neutral pi of normal serum IgA (4.5 to 6.8) (Monteiro et al, 1985). Electrostatic binding may thus play a role in mesangial IgA deposition and may be accounted for by carbohydrate composition. Glvcosvlation As mentioned above, the mesangial deposits consist predominantly of IgAl. The unique feature of this molecule is the presence o f a hinge region, which is the site of O-linked glycosylation. There is evidence, which will be discussed in detail in the following section, that in IgAN there are alterations in the usual patterns o f glycosylation, which may be of pathogenic importance. Direct interaction of IgA with mesangial matrix There is also some evidence that IgA deposition may be influenced by interactions between IgA and specific mesangial matrix components. Studies o f renal biopsy material show rebinding of IgA eluates to autologous glomeruli and to some other IgAN samples but not to normal glomeruli, suggesting that specific IgA-matrix interactions may be present (Tomino et al, 1982ii) and this too may be related to abnormal glycosylation: IgA 22

41 molecules lacking terminal sialic acid and galactose units have increased affinity for the extracellular matrix components fibronectin and type IV collagen (Kokubo et al, 1998). Mesangial IgA receptor It is now agreed that there is an IgA receptor on mesangial cells, although this has yet to be fully characterised (Monteiro & Van De Winkel, 2003). It is thought to differ from other IgA receptors thus far characterised, namely CD89 (FcaRI), polymeric Ig receptor and hepatic ASGPR. There are reports that the mesangial cell (MC) receptor may be the transferrin receptor (CD71), an Fca/jn receptor or a novel FcaR (Julian & Novak, 2004; Barratt et al, 2004). There is evidence that MCs are capable of receptor-mediated endocytosis and catabolism o f IgA, which supports the role of the MC as a significant contributor to the clearance o f mesangial IgA (Barratt et al, 2004). Why does deposited IgA cause disease? 1.10 Mechanism of glomerular injury The deposition of IgA is necessary, but not sufficient to cause glomerular injury. There is increasing realisation that genetic susceptibility is likely to be important in determining which patients go on to develop progressive glomerular disease (Barratt et al, 2004). Mesangial cell activation may result from binding of IgA to the MC receptor, leading to the production of cytokines, chemokines and growth factors (Chen et al, 1994) and to MC proliferation (Diven et al, 1998). The exposure of MC to IgA leads to up-regulation of secretion o f extracellular matrix components and the pro-fibrotic cytokine TGFp (Lopez- 23

42 Armada et al, 1996). There is also evidence that IgA has an effect on integrin expression, which is likely to play a role in mesangial remodelling (Peruzzi et al, 2000). Furthermore, aberrantly glycosylated IgA had a more profound effect, which suggests that glycosylation abnormalities may mediate both IgAl deposition and injury. While IgA is known to be poor at activating the classical complement pathway, there is evidence of local complement activation in IgAN. Data from rats indicated that multimeric IgA activated what was thought to be the alternative complement pathway (Rits et al, 1988) and in the mesangium of IgAN patients components of complement membrane attack complex have been demonstrated, co-deposited with IgA (Rauterberg et al, 1987). More recently has come the recognition that complement activation may also take place in IgAN by activation of the lectin pathway, by the binding of mannan binding protein (MBP) (Matsuda et al, 1998). There is evidence that C3 and MBP are synthesised by MC, so it is likely that once IgA is bound by MC they are capable of activating complement using endogenously generated C3 and MBP (Barratt et al, 2004) Progression It is now believed that the extent o f secondary podocyte damage determines whether resolution or progression of injury will occur (Kriz et al, 1998). Unlike MC, podocytes have little capacity to regenerate and the hallmarks of podocyte injury, namely proteinuria and segmental glomerulosclerosis, are also the hallmarks of progressive renal disease. The mechanisms of ongoing glomerular injury in IgAN are probably generic to other progressive renal disease and lie outwith the scope o f this thesis. 24

43 Pari II Glycosylation and the IgA l molecule 1.12 IgAl glycosylation The IgA molecule, like other serum and membrane proteins, and other immunoglobulins is heavily glycosylated. The functions of glycosylation include stabilising the protein structure, modifying the activity of the effector functions, shielding the protein surface from proteases and providing specific epitopes for recognition events (Rudd & Dwek, 1998). Oligosaccharides are attached to the peptide by either nitrogen- (N) or oxygen- (O) linked bonds. Unlike N-linked glycosylation, O-linked glycosylation occurs only rarely on circulating proteins, being found predominantly on membrane-bound proteins. The IgAl molecule is therefore unusual amongst circulating immunoglobulins in that it undergoes O- linked glycosylation of the hinge region (Baenziger & Komfield, 1974), a property it shares with IgD (Mellis & Baenziger, 1983) and C l inhibitor. IgA2 has no hinge region and therefore does not share this feature. In comparison to N-linked oligosaccharides, O-linked glycans are relatively simple. They are added to the protein after translation, initially by the transfer of a single N-acetyl galactosamine (GalNAc) residue in a-linkage to the hydroxyl group o f a serine (Ser) or threonine (Thr) side chain (Rudd & Dwek, 1998). Ser/Thr O GalNAc P 1,3 Gal NeuNAc NeuNAc 25

44 A terminal galactose (Gal) may be added in p 1,3 linkage with the addition of one or two sialic acid molecules (NeuNAc) (Allen, 1999i). The hinge region of the IgAl molecule is rich in serine and threonine (Mattu et al, 1998; Julian & Novak, 2004). Most authors agree there are five potential sites of glycosylation per heavy chain, ie. ten per hinge region. Studies employing protease digestion of the IgAl molecule followed by N-terminal amino acid sequencing demonstrated the exact sites of full and partial O-glycosylation in normal IgAl (Figure 1.2) (Mattu et al, 1998). These were found to be different from previous studies performed on IgAl derived from patients with myeloma. A recent study employing mass spectrometry reported a sixth GalNAc substitution on the IgAl hinge region, accounting for around 5-10% of glycoforms (Tarelli et al, 2004) but this has yet to be substantiated. Nonetheless, it is clear that IgAl exists as an array of glycoforms in which different O-glycan sites in the hinge region are occupied with a range o f oligosaccharides (Mattu et al, 1998), that is, there is microheterogeneity. CH CH2 <-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys-» i i l l i (CHO) CHO CHO CHO (CHO) Figure 1.2 The structure of the human IgA l hinge region (from Mattu et al, 1998). Between 3 and 5 O-linked glycans are attached to serine and threonine residues. In serum IgAl, serine/threonine 228,230 and 232 are occupied by the glycans, whereas threonine 225 and 236 are occupied on only a fraction of circulatory IgAl (marked in brackets). CHO.carbohydrate, Pro:proline, Val.valine, Senserine, Thr.threonine, Cys:cysteine 26

45 1.13 Abnormalities of IgA l glycosylation in IgAN The lack of a clearly defined antigenic target of the mesangial IgA in IgAN led workers to examine other features of the IgAl molecule that might explain its deposition. There is a large body of work demonstrating altered B cell N-linked glycosylation in rheumatoid arthritis (Parekh et al, 1985; Axford et al, 1987), which stimulated the investigation of glycosylation in IgAN as a potential non-immunological mechanism which might explain its deposition (Mestecky et al, 1993; Hiki, 1996i). The fact that predominantly IgAl is deposited (Valentijn et al, 1984) and that only IgAl has a hinge region (Kerr, 1990) made the hinge region the obvious place to start looking for glycosylation abnormalities. There are now many studies detailing abnormalities of glycosylation in patients with IgAN. These will be reviewed briefly, broken down by the three different approaches used to analyse the O-glycans on IgAl, namely using lectin binding to study the intact IgAl molecule; mass spectrometry of enzymatically released hinge peptides and analysis of chemically or enzymatically released glycans by size or electrical charge Lectin studies The earliest studies suggesting abnormal patterns of glycosylation in IgAN were performed using lectins. Lectins are proteins derived from plants and animals that display binding affinities for specific carbohydrate ligands (Allen, 1999ii). Abnormal lectin binding in IgAN was first described by Andre et al (1990) using the lectin jacalin; binding was found to be reduced in IgAN patients. Since then there have been many reports, from different groups around the world, of alterations in the binding of lectins in IgAN (summarised in Table 1.1 below). The majority of these studies have suggested that there is reduced terminal galactose on the hinge region of IgAl (Mestecky et al, 1993; Allen et al, 1995; Baharaki et al, 1996; Tomana et al, 1997). Discrepancies between the findings exist, 27

46 especially of the binding of jacalin: Andre and Mestecky have reported a reduction in jacalin binding but Tomino and Hiki have reported an increase (Table 1.1). Allen and colleagues used a panel of lectins, including two specific for GalNAc, Vicia villosa ( W ) and Helix aspersa (HA), the binding of which were increased in IgAN (Allen et al, 1995). Allen also used two lectins specific for Gal(31,3GalNAc, peanut agglutinin (PNA) and Amaranthus caudatus (AC) and perhaps surprisingly, the binding of these lectins was unchanged in the sera of IgAN patients. Despite the discrepancies, all authors agree that there are alterations of glycosylation of the IgAl molecule in IgAN. Some have also suggested there are abnormalities in the IgA2 molecule (Baharaki et al, 1996). In addition to discrepant results, there are other problems with the use o f lectins, which cannot provide information about precise structure or the relative distribution of O-glycans (Allen, 1999ii). Few are well characterised or evaluated. Jacalin is however fairly well evaluated, but it appears that the specificities vary depending on its source (Hiki et al, 1996i) perhaps explaining the conflicting results. Furthermore, jacalin has affinity not only for Galpl,3GalNAc, but also GalNAc, G alal,6g lc (melibiose) and to a lesser extent, galactose (Sastiy et al, 1986). The different studies reviewed have largely used different assays to examine lectin binding, which perhaps contributed to the conflicting results. Nonetheless, many groups have now reported abnormalities of lectin binding and, as suggested by Allen (1999ii) perhaps the most valid approach is the use of a panel of lectins with similar properties. 28

47 Lectin Specificity Abnormality in IgAN Reference Jacalin IgAl and IgA2 Reduced serum IgA binding Andre et al, 1990 Jacalin Gal in hinge region Decreased reactivity Mestecky et al, 1993 Jacalin IgA, IgAl and IgA2 Increased binding capacity (raised levels Tomino et al, 1995 of IgA) Vicia villosa (VV) GalNAc Increased binding Allen et al, Helix aspersa (HA) GalNAc Increased binding 1995 Amaranthus G aipi,3g alna c Unchanged caudatus (AC) Arachis hypogaea GaIpl,3GalNAc Unchanged [peanut agglutinin (PNA)] Triticum vulgaris GlcNAc Unchanged (TV) Erythrina crystagalli (ECL) D-Gal of N-linked moieties Unchanged Jacalin Sambucus nigra agglutinin (SNA) Ricinus communis agglutinin I (RCA-I) Gal P 1,3 GalNAc, GalNAc, Gal 1,6Glc > Gal NeuAca2,6Gal > NeuAca2,3Gal p i,3gal and pi,4gal Raised affinity of sera for jacalin Increased binding of IgA and IgAl Reduced binding only after neuraminidase * (hinge and Fc) Reduced Fc galactosylation Hiki et al, 1996i Baharaki et al, 1996 Erythrinia cristagalli Gaipi,4GlcNAc lectin (ECL) PNA Gaipi,3GalNAc Increased hinge region galactosylation Helix aspersa (HAA) GalNAc Increased binding * Tomana et al, Caragana arborescens (CAA) Helix pomatia (HPA) Bauhinia purpurea (BPA) GalNAc Increased binding * GalNAc Increased binding * GalNAc and Gal Increased binding * 1997 Table 1.1 Summary of the reported abnormalities of lectin binding in IgAN. The specificities and abnormalities shown are as reported by the authors. There are discrepancies as to the presumed specificities of jacalin and its binding in IgAN. Conflicting results are also found for the behaviour of ECL and PNA. Only those assays marked * were the IgA samples desialylated with neuraminidase. 29

48 Mass spectrometry Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF- MS) has been use to determine the precise molar weight of peptides and glycopeptides and has been applied to the study of the hinge regions of both serum and mesangial IgAl (Hiki et al, 1998; Hiki et al, 2001). IgAl molecules underwent digestion and the hinge region fragments separated. These were then subjected to mass spectrometry to estimate the precise molecular weights of the hinge glycopeptides. Native IgAl contained such a wide array of glycoforms that no clear peaks resulted. However using sequential exoglycosidase treatments (desialylation, degalactosylation and total deglycosylation) peaks were obtained consistent with hinge peptides carrying four or five GalNAc units and three to five galactose units, compatible with the work of Mattu et al (1998). More recent work has improved the resolution of the peaks and has also been used with sequential enzymatic digests of the IgAl hinge glycopeptides (Pouria et al, 2004; Tarelli et al, 2004). Tarelli et al (2004) have recently described a sixth GalNAc substitution on the hinge region and indicated possible locations for the GalNAc. The significance o f these findings has yet to be ascertained. IgAl from IgAN patients demonstrated increased occurrence of hinge peptides carrying less Gal or GalNAc units, although the exact structure of the hinge glycopeptide could not be elucidated (Hiki et al, 1998). The O-glycan side chains in the hinge of mesangial IgAl also consisted of significantly fewer carbohydrate side chains in IgAN versus control (Hiki et al, 2001). Tarelli and colleagues have not yet published on IgAN, but have reported reduced sialylation of the IgAl hinge in two patients with IgAl myeloma and HSP (van der Helm-van Mil et al, 2003). With the improved resolution of the peaks it appears that MALDI-ToF-MS will be increasingly used in future study of the O-glycans in IgAN. 30

49 Analysis o f free glycans by size or charge separation A further way in which glycosylation has been studied is by the analysis of free glycans, achieved by chemical or enzymatic release. These techniques have provided information on the precise structure and relative frequency of glycoforms, but clearly cannot yield information about O-glycosylation site occupancy. Iwase and colleagues (1992) used HPLC after hydrazinolysis and identified four peaks in human myeloma IgAl, but perhaps surprisingly, no GalNAc. In IgAN increased asialo-galpl,3galnac and a corresponding reduction in mono-sialylated Gaipi,3GalNAc was found (Hiki et al, 1996ii). Allen and colleagues (1999i) labelled the released glycans with a fluorophore then separated them by electrophoresis on a polyacrylamide gel. This study found a significant increase in the frequency of single GalNAc units in IgAN, which would be consistent with a reduction in terminal galactose Evidence suggestive that altered IgAl glycosylation is important in the pathogenesis of IgAN The information from the above studies is broadly conclusive that there are abnormalities in O-linked glycosylation in the hinge region of patients with IgAN; what is more difficult to prove is that these abnormalities are causally related to the clinical entity of IgAN. One interesting observation comes from a study of lectin binding in HSP (Allen et al, 1998). The same characteristic increase in W binding was found in subjects with HSP and nephritis. However in children with HSP but no evidence of renal involvement lectin binding patterns were normal. These findings are suggestive that abnormal IgAl O- glycosylation plays a role in the pathogenesis o f IgA-associated glomerular disease. 31

50 A further study from Allen and colleagues (2001) provides evidence that abnormal glycosylation is important in the mesangial deposition of IgAl in IgAN. In this small observational study nephrectomy specimens were obtained from three patients with IgAN. The O-glycosylation pattern of the eluted mesangial IgAl was more abnormal than synchronous serum samples, suggesting that abnormally glycosylated IgAl is selectively deposited in the mesangium. There is a need to be cautious in extrapolating from these data because of the small numbers involved, however they are suggestive that aberrant IgAl glycosylation is not an epiphenomenon but is pathogenic in IgAN. In a larger study of eluates from IgAN kidneys Hiki and colleagues (2001), using mass spectrometry, also found a shift of IgAl hinge glycopeptides to forms of lesser molecular weight in both mesangial and serum IgAl in IgAN compared to controls. There are also increasing numbers of in vitro studies that have provided potential mechanisms whereby abnormally glycosylated IgAl could lead to mesangial deposition and activation. Galactose-deficient IgAl has been shown to be present in high molecular mass circulating immune complexes (CIC) with IgG in significantly greater numbers in IgAN patients than controls (Tomana et al, 1997). The antigenic determinant was shown to be the Gal-deficient O-linked glycans of the hinge region by the inhibition of immune complex re-formation (Tomana et al, 1999). Such complexes might be prone to mesangial trapping and to reduced hepatic clearance related to their size (Tomana et al, 1999). Hiki and colleagues have also suggested that abnormal O-glycosylation of IgAl promotes self-aggregation demonstrating that the progressive removal o f carbohydrates from the IgAl molecule resulted in non-covalent self-aggregation and a significant increase in adhesion to extracellular matrix proteins (Kokubo et al, 1998). Thus, the under- 32

51 glycosylation of the IgAl molecule found in IgA nephropathy could be involved in the non-immunologic glomerular accumulation o f IgAl. With regard to the effects of altered O-glycosylation on the mesangium, there is in vitro evidence that abnormally glycosylated IgA up-regulates mesangial cell integrin expression, which is a marker of MC activation (Peruzzi et al, 2000) This may lead to increased cell proliferation, expression of matrix components and ultimately lead to MC contraction and altered intra-glomerular pressures. Altered glycosylation has also been shown to affect markers o f glomerular capillary repair such as vascular endothelial growth factor and nitric oxide (Amore et al, 2000) Why should IgA l glycosylation be abnormal in IgAN? Three possible explanations have been considered for the abnormal IgAl in IgAN: altered hinge amino acid sequence, alterations in circulating galactosidase or alterations in galactosyltransferase activity. Feehally and colleagues have examined all three of these possibilities and found normal amino acid sequence of the IgAl hinge region in IgAN (Greer et al, 1998). In the study of IgAl glycosylation in IgAN patients, lectin binding of C l inhibitor (Cl inh) (another circulating protein bearing O-linked glycans) indicated normal or increased galactosylation (Allen et al, 1995). Thus, there was no evidence of excess post-secretory degradation of the O-glycan chains, arguing against a defect in galactosidase function. 31,3GT activity has also been investigated by Allen et al (1997) using a novel assay. The methodology and results will be discussed in more detail in the next section and Chapter Three, but this study indicated that there was decreased galactosyltransferase activity in B cells o f patients with IgAN. 33

52 1.16 Glycosyltransferases and disease Rheumatoid arthritis Glycosylation abnormalities have been well described in a number of conditions. The best known disorder of glycosylation is rheumatoid arthritis (RA), in which there is reduced galactosylation of the Fc portion of IgG leading to an increased proportion of agalactosylated IgG (GO). This is due to reduced activity of lymphocytic pl,4galactosyltransferase, an intracellular membrane-bound enzyme which catalyses the transfer of galactose to an N-acetylglucosamine (GlcNAc) acceptor during oligosaccharide elongation (Axford et al, 1987). Treatment with sulphasalazine restores both normal enzyme activity and normal IgG galactosylation (Axford et al, 1992) The Tn polyagglutinab ility syndrome The Tn polyagglutinability syndrome (previously known as permanent mixed field polyagglutinability [PMFP]) is an acquired somatic mutation in stem cells of haematopoietic tissue caused by a defect of pl,3galactosyltransferase (pi,3gt). it is a very rare condition with no or subclinical symptoms only such as mild haemolytic anaemia, thrombocytopaenia and leucopaenia, which are related to absent terminal galactose on the Thomsen-Friedenreich (TF) antigen. The TF antigen is a membrane-bound oligosaccharide consisting of the same O-linked Gaipi,3GalNAc as is found on the hinge region of the IgAl molecule. The enzyme catalysing the conversion of the Tn antigen (GalNAc) to TF (Gaipi,3GalNAc) is a pi,3gt. In health Tn is fully substituted with galactose and, as in the IgAl system, the entity bears one or two molecules of sialic acid (Berger & Kozdrowski, 1978i). In the Tn syndrome there is selective deficiency of pi,3g T in some, but not all of the cells (Cartron et al, 1978) leading to the exposure of GalNAc, a "ciyptantigen", to which antibodies are present in most adult sera. Antibody binding leads 34

53 to clumping and to mild anaemia and thrombocytopaenia (Cartron & Nurden, 1979). After studying the pl,3gt enzyme, Thumer and colleagues (1993) found that the incomplete antigen is due to clonal repression rather than mutation or deletion of the pl,3g T gene. Treatment with an inducer of gene expression, 5-azacytidine led to de novo expression of the TF antigen Malignancy Glycosylation abnormalities have also long been recognised as a cancer-related phenomenon. The expression of cryptantigens as a consequence of incomplete or disordered glycan biosynthesis is well described (Berger et al, 1994). Tn and sialosyl Tn antigens are widely distributed on tumour cells (Springer, 1984). The activity o f a variety of glycosyltransferases, including pi,3g T was measured in colorectal cancers and surrounding tissues by King and co-workers (1994). They found that the presence of the Tn and sialosyl Tn antigens was not explained by deficient pi,3g T but was thought to be due to a defect in p i,3 N-acetylglucosaminyl (pi,3glcnac) transferase leading to loss of the GlcNAc-pl,3GalNAc-peptide structure pi,3g T activity in IgAN The studies on the Tn syndrome led to speculation that there is a similar enzyme deficiency, perhaps due to gene mutation or repression, in IgA plasma cells of patients with IgAN. A single study has been reported looking at pl,3gt in peripheral blood B cells in IgAN (Allen et al, 1997). The study measured the pl,3g T activity of the lysates of peripheral blood B cells, T cells and monocytes of IgAN patients and controls. An acceptor was prepared from degalactosylated IgAl hinge region fragments and acceptor and lysate 35

54 were incubated together with a galactose-donor, UDP-Gal. Enzyme activity was measured by changes in the binding o f the lectin W (specific for GalNAc). IgAN B cell lysates had significantly lower pl,3g T activity compared to controls, though the pi,3g T activity of T cells and monocytes were unchanged. The data suggested that the altered IgAl terminal galactosylation in IgAN results from a B cell-restricted reduction in pi,3g T activity, which may be o f pathogenic significance. Aims of the thesis Aberrant IgAl glycosylation is potentially of fundamental pathogenic significance in IgAN. Circulating IgAl in patients exhibits abnormal O-glycan glycosylation, which is also abnormal in mesangial eluates. The mechanisms of IgA deposition and the initiation of inflammation in IgAN patients may therefore relate to this feature of the IgAl molecule. Lectin studies and electrophoresis have indicated reduced terminal galactosylation of O- linked GalNAc, which is found at up to ten sites on the IgAl hinge region. Aberrant glycosylation in IgAN patients cannot be explained by altered nucleotide or amino acid sequence of the hinge region and neither is there a generalised abnormality of galactosidase activity. pl,3g T catalyses the addition of the terminal galactose and preliminary evidence exists that this enzyme may be deficient in peripheral blood B cells of patients with IgAN. The hypothesis central to this thesis was that reduced IgAl hinge region galactosylation can be explained by reduced activity of pi,3gt. As the IgAl deposited in the kidney probably derives from the bone marrow the aim of this work was to measure the activity of pi,3gt in bone marrow B cells of a cohort of patients with IgAN. The only previous study 36

55 in IgAN at the time the work was performed used a lectin-based assay lacking scientific validation. Therefore the initial part of the work was to develop an assay measuring the incorporation of radiolabelled isotope by f31,3gt, giving a direct assessment of enzyme activity. Similar assays have been used in studies of galactosylation of red cell membranes and colorectal tissues but not in B cells. The assay was developed using elements from both previously described methods and was characterised and optimised in the relevant cells and cell lines. Thereafter studies were performed of both peripheral blood B cells and of bone marrow-derived cells from patients with IgAN and healthy controls to assess whether depressed bone marrow pl,3g T activity is the explanation of altered IgAl glycosylation. The experiments were performed between January 1998 and July 1999 and are detailed in Chapters Three, Four and Five. 37

56 Chapter Two General Methods Cell culture and cell separation techniques All tissue culture procedures were carried out in a class II laminar flow cabinet (Gelman Sciences Ltd) using sterile technique, sterile plastics, solutions etc. 2.1 Density gradient centrifugation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of patients and control subjects by a modification o f the method of English and Anderson (1974). 20 ml of venous blood was taken into preservative free heparin (10 U/ml blood) and used immediately. Samples were centrifuged at 500g for 15 min, and the plasma aspirated and processed as detailed below. The cells were gently resuspended to 1.5-times the original volume in HBSS. Separate 5ml aliquots of cell suspension were then layered on to 3ml of Ficoll 400, density (Histopaque) before centrifugation (400g, 25 min, RT). The PBMC bands at the Histopaque/HBSS interface were carefully aspirated, collected together and washed twice in HBSS, centrifuging at 500g for 10 min. The PBMCs were further processed to separate the B and T lymphocytes using Dynabeads (see below) in order to measure the pi,3g T activity of these cell lysates. Typical yields of PBMC prepared by this method were 1-5 x 106 cells/ml blood and consisted of >90% lymphocytes and monocytes when measured by flow cytometry. The aspirated plasma was clotted by the addition o f 0.5g/ml protamine sulphate and 38

57 0.5 U/ml of reconstituted lyophilised bovine thrombin. After clotting had occurred centrifugation (400g, 10 minutes, RT) took place and the supernatant (serum) was aspirated and frozen. The serum was used to measure lectin binding (HA and W ) of serum IgA. 2.2 Density gradient centrifugation of bone marrow mononuclear cells Bone marrow aspirates were taken directly into syringes containing preservative-free heparin (100 U/ml bone marrow) under sterile, RNAse-free conditions. 10ml of HBSS was added to 10ml BM and vortexed hard to disrupt clumps of cells. Initial centrifugation took place (500g, 5 min, RT) then 5ml aliquots o f the cell suspension were layered gently onto Ficoll 400 as for density gradient centrifugation of PBMCs. The rest of the separation took place as for PBMCs. Typical yields o f bone marrow mononuclear cells (BMMC) prepared by this method were 6.5 x 106 cells/ml BM. The lysates of the B cell subset of the BMMC cells were used for measurement o f pi,3g T activity after further separation with Dynabeads. 2.3 Culture of human PBMCs and BMMCs After density gradient centrifugation and washing PBMCs and BMMCs were transferred into THP-1 culture medium (Appendix 1) and maintained overnight in Petri dishes under culture conditions. The maximum time in culture was 16 hours. Thereafter they were washed twice in HBSS and resuspended in HBSS in preparation for further processing. 2.4 Culture of THP-1 cells and U937 cells THP-1 and U937 are human monocyte cell lines, which express p 1,3galactosyltransferase (pl,3g T) activity. THP-1 was obtained from ECACC (No: ) and maintained in 39

58 culture medium as detailed in Appendix I. U937 (CRL 1593) was obtained from ATCC and maintained in culture medium detailed in Appendix I. All cells were incubated in a fully humidified air atmosphere containing 5% CO2 at 37 C. They were used as standards for blood and bone marrow (31,3GT activity, which can only be measured accurately in fresh tissue. The lengthy processing of samples meant that it was not possible to carry out all assays simultaneously. 2.5 Separation of cells with Dynabeads All cell separation work was performed using sterile, RNAase-free conditions as some portions of separated cells were stored for molecular biology studies or frozen for later culture studies. Cell populations were separated from peripheral blood and bone marrow buffy coat (PBMC and BMMC) by the use of a commercial kit using antibody-coated magnetisable beads (Dynabeads, Dynal (UK) Ltd.) The beads, which are 4.5pm in diameter, are coated with monoclonal antibodies to restricted cell surface proteins such as to CD 19, (a B cell restricted membrane antigen, which is widely expressed and not lost until terminal B cell differentiation into plasma cells). Thus B cells may be immobilised on a magnet after a period of incubation with buffy coat cells. Three different Dynabeads were used in this study: to isolate B cells (CD 19), T cells (CD3) and reticulocytes (CD71). The former cell type was the cell type in which the hypothesised abnormality of pl,3gt activity was expected. The latter two types were used as controls Cell preparation For B and T cell separation the buffy coat was prepared by density gradient centrifugation as described above. They were suspended in medium (Appendix 1) and left in culture conditions over night. The following day the suspended cells were gently aspirated and 40

59 collected (taking care not to disturb those that had adhered to the dish, which cytospin staining confirmed morphologically to be monocytes and were likely to cause the beads to adhere and clump). After centrifugation (250g, 10 min) they were resuspended in 3-4 ml wash buffer (Appendix 1). Bone marrow aspirate needed no preparation prior to the use of CD71 beads for the separation o f reticulocytes Dynabead preparat ion The calculated volume of beads was measured and washed in the appropriate wash buffer to remove the preservative (Appendix 1); the wash buffer used for CD71 beads was subtly different from that for the other beads used. See text in Chapters Four and Five for a description of how bead volume was reached. After gentle mixing in wash buffer the beads were immobilised on a magnet (Dynal MPC E) allowing the buffer to be aspirated. This was repeated 3 further times, after which the beads were suspended in the original volume of wash buffer Bead-cell incubation Because total cell lysate protein yield was greater when smaller incubation volumes were used the PBMCs were divided into 4 x 1.5ml Sarstedt tubes and the beads divided into 4 aliquots and added to the cells. Incubation occurred with constant gentle tilting and rotation for one hour (RT), which facilitated binding of the desired cell to the beads via the adherent antibodies Isolation and washing o f desired cell-type The cell-bead complexes were immobilised on a magnet (2 min, RT) after which the supernatant (containing all other cell types) was aspirated and collected. After removing 41

60 the tube from the magnet the cell-bead complexes were gently resuspended in wash buffer (Appendix 1) and again immobilised on the magnet. This was repeated three times, with care being taken to avoid the cells/beads from drying. The final wash was in protein-free wash buffer (HBSS/HEPES not containing FCS) to avoid any contamination of the subsequent protein assay. The cell-containing supernatant was centrifuged (230g, 8 min) and resuspended in standard wash buffer. These cells were used either for isolation of another cell type (PBMC work) or were frozen for further studies (BM) Cell lysis The cells/beads were lysed (as all other cells) using 2% Triton X 100/TBS. A volume was chosen that gave a sufficiently concentrated lysate for later use. After vigorous vortexing and pipetting the beads were immobilised on the magnet and the cell lysate was aspirated for determination o f p 1,3GT activity. 2.6 Standard cell lysate preparation THP-1 and U937 cell lines were used as standard and internal controls by which to compare pl,3gt activity of the test cells. They were aspirated from culture flasks, washed (in order to remove the protein-containing medium) and pelleted by centrifugation (160g, 5 min). A set volume o f 2% Triton X 100/TBS (Appendix 1) was added with vigorous pipetting and vortexing. The detergent broke down the cell membranes and resulted in cell lysate. The lysates were centrifuged again (6000g, 5 min, 4 C) in order to pellet the cell membranes. The lysate was aspirated for use. 2.7 Cytospin preparation and staining Cells for staining/characterisation were suspended in HBSS/PBS. A plain slide was 42

61 labelled and assembled with filter paper and specialised rubber funnel into the metal "trap". A maximum of 12 traps were loaded symmetrically in a cytospin machine (Shandon Cytospin 2, Life Sciences Ltd, Basingstoke, UK) jli1 of the cell suspension (depending on cell density) was pipetted into the funnel. The assemblies were rotated at loog for 5 minutes after vacuum-sealing the lid. The slides were allowed to air dry prior to staining. If staining was not done immediately the dried slides were wrapped in silver foil and stored at -20 C Staining with M GG Various stains were used during the course of this work and will be detailed separately in the text. To document the basic method an account o f staining with May- Grunwald/Giemsa (MGG) stain is given. MGG is useful for examining the morphology of cells. The slides were placed in a Coplin jar, in which the whole process took place. They were fixed in methanol GPR for 10 minutes after which they were washed 4 times in buffer (see Reagents) then drained. May-Grunwald (Sigma MG-500) stain was mixed 50:50 with buffer then added to the jar. The slides stood for 15 minutes and were then drained, rinsed once in buffer then Giemsa (Sigma GS-500), mixed 1:10 with buffer was added for 15 minutes. The slides were rinsed x 4 in buffer then stood in buffer for 4 minutes. After airdrying the stained slides were mounted with Xam and were examined by light microscopy Immunostaining with alkaline phosphatase The slides were fixed in equal volumes of acetone and methanol in a Coplin jar for 90 seconds and were then washed three times in TBS (see Reagents). The cells were kept moist at all times, though the surrounding slide was wiped with tissue after each final wash. Primary monoclonal antibodies were used with specificities against cell surface 43

62 markers such as CD3 and CD22. The antibody was diluted 1:50 in TBS and 50 pi was pipetted onto the cells on the slide, which were placed horizontally in a moist box. The lid was applied and the slides left for 2 hours. Unbound antibody solution was washed off with TBS, after which the second antibody, alkaline phosphatase-conjugated anti-mouse antibody was applied, at a 1:20 dilution. After a further incubation period of 2 hours the slides were thoroughly washed and the chromogenic substrate was prepared. 50pl/slide of Naphthol AS-MS phosphate (Sigma N4875) was measured to which was added lpg/ml of Levamisole (Sigma L9756) (an inhibitor of non-specific alkaline phosphatase) and a few grains of Fast Red substrate. The mixture was filtered through a dedicated funnel, applied immediately to the slides and incubated for 20 minutes. Pinkness was seen when the initial antibody had bound. In order to see cell morphology in addition to cell surface markings the slides were counterstained with Haematoxylin (Gurr Certistain, product code T, VWR International, Merck House, Poole, Dorset, BH15 1TD, England), also filtered and incubated for 2 minutes. The slides were washed in tap water and then mounted with aqueous mounting medium (Gurr Aquamount, BDH/Merck, Lutterworth, UK). 2.8 Bone marrow smears Bone marrow smears were prepared by a sample of aspirate being drawn up using a fine capillary tube, a drop placed at one end of a glass slide and spread using a slide spreader. The slides were allowed to air dry prior to staining. They were wrapped in silver foil and stored at -20 C if there was to be any delay prior to staining. Staining took place as for cytospin staining (above). 44

63 Protein chemistry and lectin binding 2.9 Protein assay Total protein concentrations of detergent-solubilised cell lysates were measured using a commercial kit (DC Protein Assay, Bio-Rad) based on the Lowry method (Lowry et al, 1951). The assay was carried out in microtitre plates according to the manufacturer s instructions, using a series of dilutions of a standard (1.39 mg/ml bovine plasma gamma globulin in TBS/2% Triton X 100) to construct standard curves. Briefly, duplicate 5pl aliquots of the standards and samples were pipetted into the wells o f a 96 well plate, followed by 25pi/well o f Reagent A (alkaline copper tartrate solution) and then 200pl/well Reagent B (Folin reagent). If the samples contained detergent, the standards were prepared in the same buffer, with detergent, ie. in the case of cell lysates as described above, the buffer used was 2% Triton X 100/TBS. If detergent was present (Triton X 100) 20pl/ml Reagent S (5-10% SDS) was added to Reagent A before use. The plates were agitated gently to mix, and allowed to stand (15-45 min, RT) for the colour to develop. The optical densities (OD) at 710 nm were then read using an automated plate reader (Titertek Multiskan Plus MKII, Labsystems, Finland). A standard curve was constructed and the sample results read from it. Samples with duplicates differing by more than 5% were rejected and repeated, as were those giving OD above or below the linear part of the standard curve; these were repeated at a more suitable dilution. The assay gave linear results over a protein range of mg/ml. 45

64 2.10 Lectin binding of serum IgAl Galactosylation of IgAl hinge region O-linked glycans from serum samples was assessed by the binding of biotinylated Vicia villosa (VV) and Helix aspersa (HA), which are specific for terminal GalNAc and whose use has been previously described (Allen et al, 1995; Tomana et al, 1997) Coating with prim ary antibody and blocking o f plates 96-well Nunc immunoplates (Life Technologies, UK) were coated overnight, at 4 C with loopl/well of rabbit anti-human IgA (Dako, UK), diluted to lopg/ml in coating buffer (Appendix I). Plates were washed 4 times using an automated plate washer (Wellwash 4; Denley, UK) with washing buffer (Appendix I) and non-specific binding sites blocked with loopl/well 2% BSA in PBS (1 hour, RT). The plates were washed prior to the addition of the serum samples Samples Serum samples were used at dilutions pre-optimised (1:100 in PBS) to ensure that the IgA binding capacity for each well was fully saturated, while minimising non-specific binding of other proteins. All standards and samples were diluted in PBS and 50pl aliquots were applied to the plates in duplicate wells. The plates were sealed and incubated overnight at 4 C Addition o f biotinylated lectins and avidin conjugated HRP Biotinylated HA and VV (both 1 mg/ml) (Vector Ltd) were diluted in PBS at 1 in 500 and 1 in 1000 to give working concentrations of 2pg/ml and lpg/ml respectively. After washing, 50pl/well of HA was added to plate 1; the same volume of VV was added to plate 2. A 90 46

65 minute incubation at RT was followed by washing and the addition of 50plAvell avidin- conjugated HRP (Vector Ltd) diluted at 1 in 2000 in PBS (2.5pg/ml) also for 90 minutes at RT Control to ensure constant level o f IgA l present A third plate was prepared as above, but instead of the addition of a lectin, anti-igal (Sigma monoclonal, 1:1000) was applied followed by HRP-conjugated anti-mouse immunoglobulin (1:1000). Development of colour was as for the lectin plates. Constant ODs after colour development indicated that the same amount of IgAl was captured from all samples and any differences in lectin binding were related to differences in glycosylation and not to different IgAl levels Colour development and quantification Colour was developed by the addition of 50pl/well o f substrate solution (Appendix I). The plates were protected from light while colour developed, and the reaction stopped by the addition of 75pl 1M H2SO4 when the colour reached an appropriate intensity. OD was read at 492 nm using an automated plate reader (Titertek Multiskan Plus MKII, Labsystems, Finland) and the means o f duplicate wells calculated. 47

66 Chapter Three Development of P 1,3 GT assay 3.1 Introduction Chapter One, Part II details the substantial body of work over the last ten years which suggests abnormalities of glycosylation of the IgAl molecule in IgAN. The majority of these studies conclude that there is reduced terminal galactose on the O-linked GalNAc moieties, which are found on the IgAl hinge region. The work of Allen et al (1997) using a functional assay of (31,3GT activity suggests that this enzyme is deficient in B cells of patients with IgAN, but not T cells nor monocytes. Previous work from Allen et al (1995) has shown reduced hinge region galactosylation in IgAN, but not of Cl inhibitor, one of the very few other serum proteins with O-linked glycosylation, which is normal or increased. The reduction in terminal galactose therefore appears to be related to a synthetic enzyme deficiency, restricted to certain cell types, rather than a circulating galactosidase. The interesting parallel of IgAN to the Tn syndrome has been drawn, in which the erythrocyte membrane-antigen TF is incomplete, lacking a terminal galactose on an O- linked GalNAc, the Tn antigen. The studies on the Tn syndrome lead to speculation that there is a similar enzyme deficiency in IgA B cells of patients with IgAN. Allen's work supports this hypothesis. 48

67 3.1.1 Methodological considerations Glycosyltransferase enzymes catalyse the reaction o f a sugar and an acceptor to produce a glycosylated substrate: enzyme Acceptor + Sugar ^ Reaction product In IgA nephropathy and the Tn syndrome the acceptor, donor, enzyme and product are as shown: pl,3g T GalNAc-O-Ser/Thr + UDP-Gal ^ Gal-pl,3-GalNAc-0-Ser/Thr The work of Allen et al (1997) is the only study of pl,3g T activity in IgAN. They used a lectin binding assay derived from the assays described earlier. The main differences of that system were the acceptor used and the method o f detection of enzyme activity. Glycosyltransferases may show exquisite specificity for donor and acceptor (Baenziger, 1994) and for this reason an acceptor derived from IgAl hinge regions was used. The preparation of degalactosylated hinge-region fragments was coated on immunoplates and cell lysates containing the (31,3GT enzyme were then added. Enzyme activity was detected by the use of a biotinylated lectin, Vicia villosa (W ), specific for O-linked GalNAc moieties. Increased galactosylation of the acceptor after incubation with the enzymecontaining-lysate were detected by reduced binding of VV, measured by optical density after a chromogenic reaction brought about by the addition of horseradish peroxidaseconjugated avidin. VV has been widely used in the demonstration o f glycosylation 49

68 abnormalities in IgAl in IgAN and so was an appropriate means of detecting changes in galactosylation. The disadvantages of this system are inherent in the use of lectins (see Chapter One, ), that is, they do not provide structural information about the glycan to which they bind and there are problems with varying specificities and cross reactivities. The data in Allen's study are consistent with reduced activity of the pl,3g T enzyme but do not prove it. What is absent is characterisation of the reaction product of the enzyme, the same glycosylated entities that have proved so difficult to characterise in the area of study of IgAl glycosylation. Unless the reaction product has been chemically analysed one cannot be certain that it is the enzyme in question functioning and whose activity is being measured. In a system where all other variables are kept constant reduced amounts of reaction product would be indicative o f reduced enzyme activity. The acceptor used in most other work investigating pi,3g T activity is the mucin, asialoovine submaxillary mucin (aosm) (Berger et al, 1978ii; Hesford et al, 1981; Thumer et al, 1992; Thumer et al, 1993 and King et al, 1994). These investigators used the incorporation of radiolabelled galactose as the means of detecting enzyme activity, which can be readily detected by gamma counting. Thus in the experimental assay the components o f the reaction are: pi,3g T aosm + UDP(14C)Gal ». OSM(l4C)Gal 50

69 The assay conditions have been well characterised in the area of study of the Tn polyagglutability syndrome. (Berger et al, 1978ii; Hesford et al, 1981; Thumer et al, 1992; Thumer et al, 1993). In addition, and importantly the product of the reaction has been clearly characterised (Berger et al, 1978ii; Hesford et al, 1981) by first cleaving the reaction product from OSM by alkaline borohydride treatment. Subsequent periodate oxidation studies were performed on the reduced disaccharide which indicated the linkage type of the product. The anomeric configuration of the products were determined with specific galactosidases (Berger et al, 1978ii). Furthermore the system was tested by incubating erythrocyte membranes from a patient with Tn syndrome with the lysates of normal erythrocytes (Hesford et al, 1981). The product displayed similar characteristics by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as normal erythrocyte membranes. The assay used by these authors therefore has been proven to measure pi,3g T activity. The study by King et al (1994) employed a similar assay using aosm as the acceptor when studying surface membrane glycosylation in malignant and non-malignant colonic tissue. Reaction products were characterised by p elimination then chromatography of the radioactive oligosaccharides, hence validating this assay. Because of the problems with lectin binding studies, namely the lack of specificity and structural information (Chapter ) this chapter describes the development of an assay to assess pi,3gt activity by adapting previously described methods, which measure the incorporation of radiolabelled substrate by pi,3g T onto an acceptor. The acceptor used by Allen et al (1997), the only previous study of pl,3g T in IgAN, has theoretical advantages over aosm in that it is the physiological acceptor for terminal 51

70 galactose in the system of interest. However there are also a number of disadvantages in its use: an assay measuring incorporation of radioisotope would require very large quantities of hinge region fragment which would be too great to be practical. The reaction product using hinge region fragments would need to be chemically characterised in order to validate its use, requiring significant extra labour and a specialist laboratory. The product of the reaction using aosm has been characterised as detailed above and thus is preferable in this respect. The literature clearly supports the use of aosm as a reliable acceptor for (31,3GT (Berger et al, 1978ii; Hesford et al, 1981; Thumer et al, 1992; Thumer et al, 1993 and King et al, 1994). For these reasons aosm was used as the acceptor and UDP(14C)galactose the donor in a pi,3g T assay system described by T Hennet (personal correspondence). The remainder of this chapter will describe the preparation of aosm and the initial assays using lysates from a number of cell lines. 3.2 Materials and Methods Preparation o f the acceptor: Asialo-Ovine Submaxillary Mucin Sialyl-OSM (sosm) (derived from homogenised sheep's submaxillary salivary glands) was kindly donated by Dr Tony Corfield. Desialylation was achieved by repeated mild acid hydrolyses at 80 C until the sialic acid content was less than 5% of the original (Corfield et al, 1985). Total sialic acid content was assessed using the orcinol ferric chloride assay (Schauer, 1978). Mild acid hydrolysis sosm was dissolved in 0.1M HC1 at a concentration of lomg/ml. A 400pl aliquot was sampled in order to assess the total sialic acid content before treatment (using the orcinol 52

71 ferric chloride assay, below). The dissolved sosm was incubated at 80 C for one hour in 2ml tubes. (The reaction is more efficient if performed in small volumes.) After cooling, the product was neutralised using 1M NaOH and was dialysed against ultrapure water at 4 C over 48 hours (4 x 21). 400pl was sampled to reassess the sialic acid content. The total remaining volume was measured and was re-acidified by the addition of 179th of the volume of 1M HC1. Three treatments were required to achieve a sialic acid content less than 5% of the original. The product was dried by lyophilisation, then stored, dry at 4 C. Orcinol ferric chloride assay The orcinol ferric chloride assay is used to measure the sialic acid content of a substance by a chromogenic reaction. An equal volume of the orcinol ferric chloride reagent (Appendix 1) was added to solution containing sosm (between 10 and loopl was used) and vortexed. The mixture was incubated at 95 C for 30 minutes then cooled for 30 minutes. Isoamyl alcohol was added ( pl depending on the reaction volume). The mixture was vortexed and centrifuged at 13,000 RPM for 10 minutes to separate the layers. 100jj.l was sampled in duplicate from the organic phase and was placed in a 96 well microtitre plate and the OD was read at 595nm on an automated plate reader (Titertek Multiskan Plus MKII, Labsystems, Finland). A standard curve was set up by performing the reaction on a series of duplicate doubling dilutions of N-neuraminic acid (Sialic acid, Sigma, Poole, UK), from 2mg/ml to 0.02mg/ml dissolved in 0.1M HC1. The assay was carried out on each known concentration of the N-neuraminic acid and the sosm solutions, sampled before and after the mild acid hydrolysis. A protein assay (Chapter 2.9) was also carried out to correct for dilution, which occurred with repeated addition of NaOH and HC1. Sialic acid content was expressed in terms of N-neuraminic acid content x 100 (arbitrary units)/mg protein in the solution. The percentage reduction in sialic acid 53

72 content was calculated. The aliquots sampled after each treatment were stored (4 C) and the assay repeated with each subsequent treatment in order to use the same standard curve and reaction conditions for the orcinol ferric chloride assay Evaluation o f the acceptor preparation Initial experiments in the development of the assay were undertaken on aosm kindly desialylated and donated by Dr Tony Corfield ( Bristol-OSM ). Once the assay was functioning (and before all of the sosm was treated) a test assay took place to compare the OSM desialylated as above ( Leicester-OSM ) with the "standard" donated by Dr Corfield. Identical results for the test samples would indicate that the home-treated OSM was accepting ( 14C)Gal appropriately and was therefore satisfactorily desialylated Selection o f a standard cell lysate pl,3g T activity is best measured in fresh tissue as there is experimental evidence to suggest that freezing reduces enzyme activity (Pearce et al, 1996) including specific data on galactosyltransferases (Fraser & Mookeijea, 1976). Preliminary observations performed in our laboratory supported these findings (Allen, unpublished observations). Because the intended tissue was human bone marrow it was not possible to collect all samples and perform the assay on one day, which would allow direct comparison. Therefore in order to compare the pi,3g T activity of test samples a cell line was required, the lysate of which exhibited pl,3gt activity and which could therefore be used as a standard against which test samples could be measured. Three human cell lines were evaluated: NCI H929 (a B cell clone from a patient with myeloma), THP-1 (a monocyte line from a child with leukaemia) and U937 (a more differentiated monocyte line). Each of these cell lines had p 1,3GT activity. The latter two grew consistently and well under the conditions described 54

73 in Chapter 2.1. The relative activity of these two cell lysates were evaluated for constancy, in which case they would be suitable as "standard" and "standard control" for subsequent assays of test samples. The activity of the standard cell lysate was assigned arbitrary units (AU), by which all other samples were compared. Cell lysates were prepared as detailed in Chapter 2.5 and the total protein content of all lysate samples was measured by the Bio- Rad DC Protein Assay, as described in Chapter pi,3g T assay Preparation of samples, standards and controls The cells were lysed with 2% Triton X 100/TBS and were prepared as detailed in Chapter 2.5. The lysate was aspirated after pelleting of the cell membranes. A standard curve of U937 cell lysate activity was set up each time the assay was performed by carrying out the reaction on 8 different dilutions of lysate. A standard control of THP-1 cell lysate was also performed with each reaction to demonstrate that the standard was constant in its activity. This was performed at 3 different dilutions. Each lysate sample was run in duplicate and as a control a "blank" was also run, whereby the lysate and the substrate were incubated separately; the substrate was only added to the enzyme after quenching o f the reaction. Reaction mixture The assay was carried out in 75pl volumes. 50pl of substrate solution was added to 25pi of cell lysate in 1.5ml Sarstedt tubes at 37 C and was then vortexed. The substrate mixture contained 2mg/ml aosm, 20mM MnCB, 1% Triton X 100, 0.25mM UDP-Gal and lopl/ml UDP(14C)Gal made up in 50mM Tris ph 7.0 (see Reagents). After one hour of incubation at 37 C the reaction was quenched by the addition of 500pl of phosphotungstic acid/trichloroacetic acid (5%/l 5% v/v) (PTA/TCA). Timing was meticulous such that substrate was added to lysate in strict rotation at intervals o f 2 0 seconds and the quenching 55

74 of the reaction followed the same order and interval. After cooling (4 C) for 30 minutes the mixture was centrifuged (14,000 g, 5 minutes, 4 C) and the supernatant poured off. The product was washed twice in 1ml of 95% ethanol with repeat vortexing and centrifugation between each wash. Finally the drained reaction product was dissolved in 500pl of 0.2M KOH (overnight, 37 C). Detection of pi,3g T activity The dissolved reaction products were vortexed and transferred to scintillation vials. To each was added 2ml of Ecoscint A scintillation fluid (National Diagnostics ref. LS-273). The mixture was vortexed and left to stand 12 to 24 hours while chemiluminescence (which would have interfered with the beta counter) faded. Measurements were also made of 50pl of substrate, which had undergone the incubation without the presence of cell lysate and of scintillation fluid alone. Scintillation counting took place the following day in a beta counter (LKB 1219 Rack Beta Liquid Scintillation Counter) with 226Ra external standardisation and quench correction. The results were expressed in disintegrations per minute (DPM). Calculation of p1,3gt activity in the test cell lysate samples The means of duplicate samples were calculated and the value of the "blank" was subtracted to correct for background radioactivity trapped in the precipitated protein. The protein content of each lysate was calculated in pg (globulin-equivalent) per reaction. Each pg of U937 protein was arbitrarily assigned an enzyme activity of 1 arbitrary unit (AU). Background counts were subtracted and the standard curve was constructed by plotting the DPM against AU. The enzyme activity of both the test and the standard control lysates were then read from the standard curve and expressed as AU/pg total lysate protein. 56

75 3.2.5 Evalnation o f (31,3GT assay Demonstration that cell lysates catalyse the incorporation of ( 4C)Gal A series of doubling dilutions of the standard lysate protein was used in the assay procedure outlined above in order to: demonstrate that cell lysate contained enzyme activity with the ability to cause incorporation o f isotope; demonstrate that this activity was dose dependent with respect to protein concentration o f lysate; find a suitable range of dilutions for construction of standard curve in subsequent assays; find suitable protein concentrations of the standard control lysate to ensure that activity o f at least 2 dilutions falls onto the standard curve; find a suitable protein concentration of test samples at which to measure the enzyme activity in subsequent assays. In the light of these experiments the remainder of the assay evaluations were carried out using approximately doubling dilutions of the standard (U937) lysate consisting of 8 dilutions ranging from 0.1 to lomg/ml. The standard control (THP-1) lysate was used at a concentration between 1 and 5mg/ml and the test lysate samples at a concentration of between 0.4 and 3mg/ml. Necessity for a glycosidase inhibitor Dimercaptol propan-l-ol (DMP) is a glycosidase inhibitor (to prevent the breakdown of the enzyme product), which is used in assays performed on colorectal tissue at a concentration of 1.3mM (King et al, 1994) but not in assays on erythrocyte membranes (Berger et al, 57

76 1978ii). An evaluation was performed as to whether this substance would increase the sensitivity of the pl,3gt assay. A preliminary assay (data not shown) suggested that 5pl/ml DMP increased the sensitivity of the system. Two parallel standard curves were therefore set up at six different dilutions o f lysate protein. Two substrate solutions were prepared, one containing 5pl/ml DMP and the other containing none. Effect of halving reaction volumes The objective of the work was to measure (31,3GT activity in the bone marrow B cells of patients with IgAN. These were fractionated using Dynabeads as detailed in Chapter 2.5. The protein yield from these cell lysates was at the bottom of the range needed to assess enzyme activity, ie. the bottom of the standard curve. An evaluation was therefore performed of the effect of halving the reaction volumes (both substrate and cell lysate), which would, if successful, effectively double the concentration of protein achieved (only half the volume of TBS/TritonX 100 would be used to lyse the cells). Two experiments were run in parallel using 4 concentrations of standard lysate, 2 concentrations of control lysate and 2 concentrations of test lysate (PBMCs). For the standard conditions 50pl of substrate solution was added to 25pl of lysate. For the reduced-volume experiments the same solutions were used but 25pl o f substrate was added to 12.5jnl of lysate. Reproducibility of the p1,3gt assay Intra-assay coefficients of variation (CVs) were calculated for the assay using eleven sets of duplicate reactions of THP-1 lysate measured against the standard curve of U937 lysate and the assay carried out as described (3.2.4). The inter-assay CV was calculated for the THP-1 lysate activity in each assay over the period of a series of 15 experiments occurring over a period of 3 months. 58

77 3.3 Results Eval uation o f acceptor A sialic acid standard curve (used in the orcinol ferric chloride assay) is shown in Figure 3.1 and the effect of mild acid hydrolysis on the OD550 and sialic acid content of the progressively desialylated OSM samples is shown in Table o l o Sialic acid content (mg/ml) Figure 3.1 Orcinol-ferric chloride assay: typical standard curve. A standard curve was constructed by a series of doubling dilutions of a commercial preparation of sialic acid, which underwent the orcinol-ferric chloride assay. Results were expressed in O D The sialic acid content of test samples could be read from the curve. The arrow represents the sialic acid content of OSM after desialylation, which could not be read from the standard curve as it is below the straight part of the line. 59

78 Sample (protein content mg/ml) OD550 minus Sialic acid Sialic acid background content content (mg/ml) corrected for dilution (mg/ml) s-osm (0.64) % reduction in SA content [cumulative] OSM-1 (1.8) % [29%] OSM-2 (1.8) % [9%] OSM-3 (1.8) 8 Table 3.1 The reduction in sialic acid content of sialyl-osm (s-osm) as it underwent progressive desialylation. OSM-1 : after one hydrolysis, OSM-2 after two hydrolyses etc. Protein concentration (adjusted) is given in parentheses in 'Sample' column. Sialic acid content o f sosm (0.64) was multiplied by 1.8/0.64 to correct for initial dilution. Each treatment resulted in a reduction in sialic acid content of about 30%. In this assay the sialic acid content of OSM-3 was at the bottom of the standard curve (arrow), so the exact percentage reduction could not be assessed. If a further reduction of sialic acid content of c.50% occurred on a third treatment a final sialic acid content of less than 5% of the original would be achieved. As this has not been demonstrated a functional assessment of the desialylated OSM was carried out alongside aosm to ensure that it behaved as a satisfactory acceptor for donor (14C)galactose, the results of which are detailed in the next section. 6 0

79 3.3.2 Evaluation o f the acceptor preparation The results of the pl,3g T assay using the Bristol aosm and the in-house preparation (Leicester aosm) are shown in Figure 3.2. There was incorporation of isotope by both acceptors, indicating that the treated aosm was satisfactory. The two curves move in parallel, with the Leicester-treated aosm revealing slightly lower counts. The enzyme activity of the control lysate revealed equivalent activity, 1.2 and 1.16 AU/pg for Leicester and Bristol OSM respectively. O) "I 3000 n. ^ 2000 Q. Q 1000 Leics OSM * Brist OSM 1 10 AU (THP-1) Figure 3.2 pl,3gt assay to compare the functioning of home-desialylated OSM (Leicester) with the standard donated by Dr Corfield (Bristol). A standard curve was constructed by measuring (31,3GT activity in 4 concentrations of THP-1 cell lysates. Enzyme activity of a further cell line (NCI H929) was measured and expressed in terms o f arbitrary units (AU) equivalent to THP-1 lysate protein (pg). 61

80 3.3.3 Evaluation o f the fil,3g T assay Demonstration that cell lysates catalyse the incorporation o f(u C)Gal The results of radioisotope incorporation after incubation of substrate with a series of dilutions of the standard lysate are shown in Table 3.2. The standard lysate (U937) was assigned an enzyme activity of 1 AU/pg lysate protein. Figure 3.3 shows a typical standard curve constructed by plotting enzyme activity of the lysate dilutions in AU against DPMminus-background. 62

81 Lysate protein (mg/ml) Protein/assay (Pg) pi,3gt activity (AU) DPM (mean) DPM minus background U U12B 66 U U8B 64 U U4B 52 U U2B 60 U U1B 50 U U0.5B 55 U U0.25B 55 S Table 3.2 Construction of standard curve for pl,3g T assay using U937 cell lysate. The standard curve was drawn by plotting AU against DPM-minus- background. U8 : denotes U937 lysate at a protein concentration of 8 mg/ml. B : blank reaction, in which the substrate solution was added after 1 hour's incubation and quenching of the reaction with PTA/TCA. S : pure substrate. In this reaction c. 28% of substrate was incorporated in the reaction containing maximum cell lysate protein, judged by DPM incorporation. Protein/assay is the absolute quantity of protein per reaction. In the development work 25pl of lysate was used. In the bone marrow studies only 12.5pi were used. U937 lysate was attributed an arbitrary activity of 1 arbitrary unit (AU)/pg of protein. DPM : disintegrations per minute. The values shown are means of the duplicates. The DPM of the blank reactions were subtracted from the active reactions to give the DPM-minus-background. 63

82 4000 i c 3000 O ) P1,3GT activity (AU) Figure 3.3 pi,3g T activity of U937 lysate: typical standard curve. The arrow shows the part of the curve, above which the test lysate results may be read. The value for each test sample was corrected for the protein concentration (jug per reaction) to express the results as AU/pg. Between 15 and 30% of donor substrate was incorporated by the reaction containing maximum cell lysate protein. The variability is explained by variations in concentration of standard cell lysate protein. The activity of the lysates showed an exponential doseresponse pattern, which was near linear at a protein concentration greater than 0.75mg/ml, i.e. AU greater than about 17. The optimum range for a standard curve for use in subsequent assays was between 0.25 and lomg/ml of U937 lysate protein. The standard control (THP-1) lysate was used at concentrations between 1 and 5mg/ml and other cell lysates were used at concentrations between 0.4 and 3mg/ml. 64

83 Necessity for a glycosidase inhibitor The results of the experiment to assess the necessity of adding DMP to the substrate are shown in Figure 3.4, which used 6 different concentrations of lysate. There is no difference between the curves. As a result DMP was not used in subsequent assays i with DMP a no DMP AU (THP-1) Figure 3.4 The effect of using the glycosidase inhibitor dimercaptopropan-l-ol (DMP) 5pl/ml on THP-1 lysate pl,3g T activity compared to none. There is no difference in the sensitivity of the assay with DMP. 65

84 Effect of halving reaction volumes Figure 3.5 shows the results of the experiment to assess the effect o f halving the reaction volumes. Ideally each curve should have had more than 4 points and the half-volume curve should have had an AU value of around 100. However in order to achieve this a more concentrated solution of lysate would have been needed. From the curves plotted the pi,3g T activity of the control (THP-1) lysate was 0.5 and 0.4 AU/pg for full- and halfvolume reactions respectively and for the test lysate (PBMC) 0.58 and 0.6 AU/pg respectively. Although it would have been preferable for the values for THP-1 lysate activity to be more similar, the values for the PBMC lysate were satisfactory. On the basis of this experiment the reaction volumes were halved for all the bone marrow assays in order to use the scarce protein most effectively o. [standard volume * : 1/2 volume P1,3GT activity (AU) Figure 3.5 The effect of halving the reaction volumes on pl,3g T activity. Two standard curves were prepared and J31,3GT activity o f a test lysate at both volumes was measured. Regression analysis showed that the slopes for the two lines, after loglo transformation of 31,3GT activity were for full volume and for the half volume reactions. 66

85 Reproducibility of the 01,3GT assay Intra-assay and inter-assay coefficient o f variance (CV)s were calculated from: CV = standard deviation x 100% mean The intra-assay CV calculated from 11 sets of duplicate reactions o f the same lysate was 7%, while the inter-assay CV, calculated from the relative value of THP-1 to U937 lysates over the course of a series o f experiments was 16%. The data is shown in Table

86 Intra-assay CV Inter-assay CV Replicates pl,3g T activity Replicates pl,3g T activity (assayed on a single occasion) AU/pg (mean of 2 duplicate reactions) (assayed on 15 occasions over consecutive assays) AU/pg (mean of 2 duplicate reactions) Mean 1H SD CV% 7% Mean SD CV% 16% Table 3.3 Intra-assay and inter-assay coefficients of variation (CV) of pl,3gt assay of THP-1 activity in AU/pg protein. 68

87 3.4 Conclusions The set of experiments in this chapter concerned the development and evaluation of an assay system to detect and quantify the activity of 31,3GT in lysates of PBMCs and three monocyte/lymphocyte cell lines. This assay had not previously been used in our laboratory. An acceptor was prepared by the desialylation of a mucin from sheep's submaxillary salivary glands, ovine submaxillary mucin. The degree of desialylation was measured using the orcinol ferric chloride assay and the acceptor was tested functionally by comparing its use in the assay system alongside the donated aosm. Radiolabelled ( 14C)galactose was incorporated onto the aosm by incubation with fresh cell lysates, which was measured by scintillation counting. The amount of isotope incorporated was proportional to the protein concentration at a concentration greater than 0.75mg/ml of U937 lysate. Similar activity was demonstrated in lysates of THP-1 and NCI H929 cell lines and fresh PBMCs. An alternative acceptor was evaluated and found not to incorporate ( 14C)Gal (data not shown); this finding is consistent with the known marked specificity of glycosyltransferases for both substrate and acceptor. The addition of a glycosidase inhibitor was assessed and found not to increase the sensitivity of the pl,3g T activity in cell lysates. The final composition of the substrate solution is shown in Table 3.4. This substrate solution was used for all other 31,3GT assays described in this thesis.

88 Ingredients mmol mg/ml 50 mm Tris, ph 7.0 MnCl UDP-Gal aosm 2 Triton X 100 1% vol/vol lopl U DP('4C)Gal 250nCi/ml lopl Cells lysed : 2% (vol/vol) Triton X 100 Table 3.4 Final composition of substrate solution. Components are shown in molar quantities and mass. 3.5 Discussion The 31,3GT assay described by Berger et al (1978ii) is a fully validated assay, known to measure the incorporation of galactose onto GalNAc-O-Ser/Thr. Although not previously used in the investigation of the glycosylation abnormalities in the field o f IgAN, the initial assays described in this chapter suggest that this assay is highly applicable to this area. The assay is carried out on fresh cell lysates and produces reproducible results when carried out on standard and control lysates derived from cell lines. This yields the opportunity to perform comparative assays over a period o f time. 70

89 The advantages of the system are that the product has been characterised (Hesford et al, 1981; King et al, 1994) and it is thus scientifically validated. While the molecular biology of galactosyltransferases is increasingly understood and a number of different GTs had been identified (Hennet et al, 1998; Zhou et al, 1999) this had not (at the time of the study) resulted in a molecular method for detecting mrna or protein of the pl,3g T of interest. Two forms of core 1 pl,3g T have now been cloned (Ju et al, 2002ii; Kudo et al, 2002) and will be discussed in Chapter Six. The assay system described was therefore the best characterised and accepted at the time of the study. The disadvantages and limitations have also largely been alluded to; Allen's method was simple, cheap, and convenient. It used only small volumes of enzyme/lysate and probably employed a more physiological acceptor. It is however possible that isolated hinge region peptides are able to accept more O-linked glycans than the intact IgAl molecule because of greater access of enzyme to the peripheral serines and threonines, which might otherwise be blocked by the Fc portion of the molecule (Iwasaki et al, 2003). A potential criticism of using this assay in lymphoid cells is that the reaction product has not been characterised and unless this is done for each experimental setting in which the assay is used, we cannot be certain that the radiolabelled sugar has been incorporated in a P 1,3-linkage. If incorporation were into any other site we would not be measuring pl,3gt activity. The characterisation of product however was not practicable in that this is done on large volumes of product and the analysis is performed in specialist laboratories. Given the very close similarities to the Tn syndrome (in terms of acceptor, donor glycan and enzyme) it was felt that product analysis in our laboratory was neither necessary nor feasible. 71

90 Because of the requirement of the assay to be performed on fresh tissue, it was not possible to perform direct comparisons between samples, resulting in the need to perform comparisons against a standard. This increased the work and the possibility of errors. Cell lines are fragile and prone to infection. The standard nature of the "standard" was clearly an absolute requirement of such a system if comparisons were to be made. In order to overcome these problems all cells were kept in one incubator. Once weekly all cells were combined, centrifuged and resuspended in fresh medium. Meticulous attention to antiseptic practice was observed and all flasks were closely inspected for infection or cells that were growing poorly. The cells were sampled and assays performed on the same days in relation to feeding days in order to keep activity/metabolism of the cells constant. The "standard control" was an indicator of the system's functioning and therefore provides an internal measure o f constancy and validity o f the comparisons made across time. (31,3GT activity was found widely in monocyte and lymphocyte cell lines and in PBMC lysate. Only 10% of PBMCs are B cells, of which a variable proportion are IgAl B cells. This enzyme is therefore clearly not IgAl-specific. The subsequent experiments using this assay all employ methods for fractionating the cells in order to focus the study on the cells of interest. The subsequent experiments using this assay are described in the following chapters with the details o f cell fractionation. 72

91 Chapter Four (31,3GT activity in peripheral blood lymphocytes of patients and controls 4.1 Introduction Thus far this thesis has reviewed glycosylation abnormalities of the IgAl molecule in IgAN and outlined reasons for considering that these may be of pathogenic importance. It has discussed the only previous study in the field which found decreased pi,3g T enzyme activity of B cells of patients with IgAN (Allen et al, 1997) and has discussed some of the potential limitations of the use of lectins in this study and generally. The work on the Tn syndrome has been reviewed both for the reason that there is clonal suppression of pi,3gt, which may be of relevance in IgAN and because of the extensive experience of these investigators in measuring galactosyltransferase activity (Berger et al, 1978ii; Hesford et al, 1981; Thumer et al, 1992; Thumer et al, 1993). The development and validation of an alternative method for measuring f31,3gt activity has been described in order to test the hypothesis that altered IgAl hinge glycosylation in IgAN is caused by reduced pl,3g T activity. The initial intention was to duplicate and confirm reduced pl,3g T activity in peripheral blood B cells of IgAN patients as found by Allen et al (1997). The ultimate plan was to study, for the first time, pl,3g T activity in bone marrow, the likely origin of the IgAl deposited in the glomerular mesangium. This chapter will document the measurement of pi,3g T activity in subsets of cells derived from blood as a preliminary series to ensure that the assay system was optimised and to confirm the previous findings. 73

92 The results of individual (M,3GT activity were correlated to the galactosylation of the O- linked glycans of serum IgAl from the same subjects, assessed by the binding of biotinylated W and HA lectins, (both specific for GalNAc). A negative correlation would be expected if reduced B cell f31,3gt activity were responsible for reduced galactosylation as found by Allen et al (1997). 4.2 Subjects and Methods Subjects Ten patients with IgAN agreed to take part in this study. They were identified from the departmental database of patients with biopsy-proven IgAN. Only those with a normal serum creatinine at last clinic review were approached. In order to keep the study group as homogenous as possible, non-caucasian patients and those with proteinuria greater than 2g/24h were excluded, as were patients with other systemic conditions such as diabetes and those with Henoch Schonlein purpura. A letter explaining the bone marrow study and an invitation to participate was sent by post to the patients with a tear-off slip and a stamped self-addressed envelope. A follow-up telephone call was made to those who failed to reply. Initially 15 patients diagnosed in the previous three years were approached, of whom seven replied favourably. Five o f these patients agreed to venesection prior to the BM study. Further letters were sent to 16 patients diagnosed between three and five years earlier, of whom six replied positively and four were venesected. One other patient, previously accommodating with departmental research was approached and agreed to the procedure. The control samples were drawn from clinical, laboratory and support staff, selected by 74

93 age and sex matching. All were well with no renal, systemic or inflammatory diseases. 100 ml venous blood was drawn from ten patients with biopsy proven IgAN (seven male, median age 40 years, range 28 to 61 years). All patients had a normal serum creatinine, none had nephrotic range proteinuria and the only permitted medications were antihypertensive agents. Blood was also taken from ten controls (also seven male, median age 37 years, range 33 to 60 years). The studies had the approval of Leicestershire Ethics Committee Samples and cell lysates/controls The blood was taken into syringes containing 10 U/ml blood of preservative-free heparin, and PBMCs were separated as described in Chapter 2.1. The serum was separated (Chapter 2.1) and was frozen in aliquots at -20 C for subsequent measurement of IgAl lectin binding. After the PBMCs were separated into mononuclear cell fractions, they were washed and resuspended in THP-1 medium (Appendix 1) and were maintained under culture conditions overnight. The next day the cells were sequentially fractionated into T and B cell populations using positive selection with anti-cd 3 and anti-cd 19-conjugated magnetic beads respectively (Dynabeads, Dynal Ltd, Wirral, UK) as detailed in Chapter 2.5. The cells for "standard" and "standard control" lysates were prepared as described (Chapter 2.6). Protein content of all lysate samples was measured as described in Chapter 2.9). The lysates were then ready for the measurement o f (31,3GT activity Immunohistochemistry In order to be sure that the magnetic beads were selecting the desired cells, that the selected population was pure and that the desired cells were not being lost 75

94 immunohistochemistry was performed on cytospin preparations of the cells of interest (Chapter 2.7). A useful feature of overnight incubation appeared to be that a population of adherent cells, believed to be monocytes, stuck to the base of the culture dishes. They remained adherent unless cooled (4 C) then flushed forcefully with cold PBS. Their adherent properties meant that if present in the Dynabead incubations they could cause clumping of cells with the desired cells and beads causing contamination; important as monocytes are known to have pl,3g T activity (Allen et al, 1997). Cytospins were prepared of the immediate PBMC sample; of PBMCs after overnight incubation and loss of adherent cells; of the adherent cells; of the cells selected by CD3 beads; of the cells selected by CD 19 beads and of the cells that were not selected by either magnetic bead type. The cytospins were stained using monoclonal primary antibodies to cell surface markers and an alkaline phosphatase-labelled anti-mouse secondary antibody as detailed in Chapter Colour was developed with a chromogenic substrate. The antibodies used were anti- CD 3, anti-cd 22, anti-cd 14 and anti-dr (against T cells, B cells, monocytes and HLA Class II respectively). Haematoxylin was used as a counter stain to enable recognition of cell morphology. Cells were counted under light microscopy on a haemocytometer slide with the aid of a cell counter. The total numbers of haematoxylin-stained cells were counted and the proportions that were T cells, B cells and monocytes identified by their staining characteristics and morphology Determination o f optimal volume o f Dynabeads From the results in Chapter Three it can be seen that pl,3g T activity increases with the protein concentration. It was clearly necessary to maximise the protein yield from the 76

95 fractionated cells. This was particularly relevant to B cell protein as B cells are less abundant in peripheral blood than T cells. An evaluation was performed using different volumes of Dynabeads to assess the maximum B cell protein yield. PBMCs were prepared from 60ml of blood, which were divided into 4 equal aliquots after 0.5ml was aspirated for cell counting. A preliminary assay was suggestive that 50pl o f CD19 beads/15ml blood maximised the yield, however 50jxl was the largest volume used and it was therefore unclear if increasing the volume further would increase the yield. The volumes of Dynabeads used were 25jul, 50pl, 75pl and loojal per 15ml blood. B cell separation with the beads took place as previously described (Chapter 2.5). After the final wash 300pl aliquots were removed from each concentration for further cell counting. The cells were lysed with 50pl TBS/Triton X 100, the beads were immobilised and the cell lysate was aspirated. Protein concentration was measured as previously described. The assay was performed in duplicate with PBMCs from two healthy volunteers. As a result of this assay 50pl CD 19 beads were used per 15ml of blood, which equated to 330pl for 100ml blood p l,3 G T activity o f peripheral lymphocytes The assays (as detailed in Chapter 3.2.4) were carried out on 10 occasions over a period of 6 weeks; on each occasion samples were used from one IgAN patient and one sex- and age-matched control. A U937 standard curve was prepared and lysates of 3 concentrations of THP-1 were used as internal controls. For each subject assays were performed on one concentration of B cell lysate and 2 concentrations of T cell lysate, which had been prepared using the manufacturer's recommended volume o f CD3 beads, namely, 80pl Reproducib il ity o f the p i, 3 G T assay The validity of comparisons made between assays run on different days is dependent on 77

96 the constancy of the relationship between the activities of the internal standard (THP-1 cell lysate) and the standard lysate (U937). The inter-assay CV was therefore calculated for the THP-1 lysate activity in each assay over the series of 10 experiments Vicia villosa and Helix aspersa lectin binding o f serum o f IgA l Measurement of lectin binding was performed as described in Chapter The results were expressed as OD 492nm, which are relative units and meant that results from assays performed at different times could not be directly compared with one another. For this reason all samples were run in a single assay on a single immunoplate Statistical analysis (31,3GT activities of T cell and B cell lysates in IgAN and controls were compared by unpaired Student Mests, as were lectin binding results of serum IgA. Linear regression analysis was carried out to investigate the relationships between (31,3GT activity of each cell type and lectin binding of serum IgA from the same subject in IgAN and controls. Linear regression analysis was also performed to investigate the relationship between (31,3GT activity of test and control cell (THP-1) lysates because of concern about a general drift upwards of values of pl,3g T activity. 78

97 4.3 Results Immunoh is toe hem is try The proportions o f cells at the different stages o f cell separation are shown in Table 4.1. T cells Monocytes B cells DR+ve PBMC 6 6 % (5.4) 29% (4.4) 9% (1.8) 26% (5.3) After O/N incub11 72% (1.6) 7% (0.8) 11% (3.1) 20% (2.7) Adherent cells 20% (2.9) 63% 6.3) 7% 1.3) 76% (5.0) Post-CD 19 beads 92% (4.9) 92% (4.9) Excluded cells 74% (2.8) 11% (1.3) 10% (1.9) 18% ( 1.0 ) Post-CD 3 beads 94% (5.7) Table 4.1 The percentage of cell types by immunohistochemistry at various stages of cell separation prior to measuring pi,3gt activity. T cells, monocytes and B cells were characterised by their positivity for the cell surface markers, CD 3, CD 14 and mixed CD 19 and 22 and morphological characteristics. Anti-DR is a cell surface marker for HLA Class II cells, which is expressed by monocytes and B cells amongst others. The figures are means for multiple cell counts on up to 5 different cytospin preparations. The standard error o f the mean is shown in parentheses. The table demonstrates that the initial predominant cells were T cells; about 10% of the cells were B cells. The monocyte fraction was reduced by overnight incubation, which is also reflected in the enrichment o f monocytes in the adherent cell population. The B and T cell populations were each around 90% pure after magnetic bead isolation. The stained cytocentrifuge images are shown in Figures 4.1, 4.2 and

98 I * % Figure 4.1 B cells separated from peripheral blood by density gradient centrifugation and antibody-conjugated magnetic beads. Figure 4.1a (above) Cells positively stained for HLA-DR. Figure 4.1b (below) Cells exhibiting no staining for CD3. 8 0

99 Figure 4.2 T cells before and after separation. Figure 4.2a (above) T cells in PBMC preparation stained for CD3. Figure 4.2b (below) After separation by density gradient centrifugation and antibody-conjugated magnetic beads, stained for HLA-DR (negative). 81

100 Figure 4.3 Monocytes before and after overnight incubation. Figure 4.3a (above) Monocytes in PBMC preparation stained for CD 14. Figure 4.3b (below) After overnight incubation the adherent cells were cooled and removed by flushing with ice-cold PBS. Stained for CD

101 4.3.2 Determination o f optimal vol ume o f Dynabeads The results from the experiments to maximise the yield of B cell lysate protein are shown in Tables 4.2 and 4.3. In the case of each subject the maximum protein yield was achieved by using 50pl CD 19 Dynabeads/15ml blood This is therefore the volume o f B cell beads that were used throughout this series of experiments. 330pl beads were used for 100ml blood. Subject Bead volume (HO Total volume (ml) Volume removed (ml) Cell count/ml Total cell count A x x x x x x x x 105 B x x x x x x 10s x x 105 Table 4.2 B cell counts achieved using different volumes of antibody-conjugated magnetic beads for subjects A and B. 83

102 Subject Bead volume (HO B cell protein (M-g/ml) Lysis volume (Hi) B cell protein yield (Hg) Protein yield/15ml blood (Hg) Total achievable protein (Hg) A B Table 4.3 Maximum B cell protein yield using a range of volumes of CD19- magnetic beads. In each case one fifth of the volume had been removed so the protein yield per 15 ml blood is 1.25 x the measured yield. The total achievable protein is the calculated yield if the corresponding volume of beads were used on 60 ml of blood (31,3GT activity o f peripheral lymphocytes Table 4.4 shows pi,3g T activity for patient and control B and T cell lysates. No statistically significant differences were found between patient and control B or T cell lysate activity using Student's unpaired t test. There was a statistically significant difference between patient B and T cell pi,3g T activity (p=0.022) but not for controls, the importance o f which is unclear. 84

103 Experim ent THP-1 B cell pl,3g T activity T cell pl,3g T Number activity (AU/pg) activity (AU/pg) (AU/pg) Patient Control Patient Control Mean SD CV 32% sem P Table 4.4 pi,3g T activity of patient and control peripheral blood B and T cell lysates (AU/pg) and the activity of THP-1 cell lysates. No data exist for T cell activity for experiment 1. The CV of THP-1 activity was 32%. No statistically significant differences were found by Student's unpaired t tests between patient and control B or T cells. Comparison of the pi,3gt activity in the B cells and T cells showed a p value of for the patients and for the controls. 85

104 4.3.4 Reproducibility o f the fil,3 G T assay The raw data for THP-1 {31,3GT activity during the six-week series o f experiments are shown in Table 4.4. The CV was poor at 32% and will be discussed further in the Discussion Relationship between the f31,3gt activity o f test cell- and THP-1 lysates The correlation coefficients and p values from linear regression analysis performed to examine the relationship between (31,3GT activity of patient and control cell lysates and that of THP-1 lysate are shown in Table 4.5. There were significant positive relationships between the activities of THP-1 lysate activity and all test samples with the exception of patient B cell (31,3GT activity. pi,3g T activity r value p value Patient B cell vs. THP Control B cell vs. THP * Patient T cell vs. THP * Control T cell vs. THP * Table 4.5 The relationships between test cell pl,3gt activity and that of THP-1 lysates. There were significant correlations between control B cell-, patient T cell- and control T cell-lysates and THP-1 lysate activity. * denotes a significant correlation 86

105 4.3.6 HA and W lectin binding of serum of IgA 1 The binding of HA and W lectins are shown in Table 4.6. Contrary to expectations and previous findings there were no statistically significant differences between patient and control lectin binding. Linear regression analysis of B cell (31,3GT activity and HA and W binding revealed no significant correlation. Subject HA lectin binding W lectin binding num ber (OD) (OD) Patient Control Patient Control mean sem P Table 4.6 HA and W lectin binding of patient and control serum IgAl expressed as OD at 492nm. Patients and controls were matched for age and sex. There was a technical error in processing serum o f patient number 7 so no data were generated. No statistically significant differences were found by Student's unpaired t tests between patient and control HA or VV lectin binding. 87

106 4.4 Conclusions The series of experiments described in this chapter were the first using the pl,3g T assay, developed and evaluated in Chapter Three. The assay was performed on peripheral B and T cell lysates from patients with IgAN and age- and sex-matched controls. The strategy employed to separate subtypes of cells using antibody-conjugated magnetic beads was documented to be effective using immunohistochemistry and morphometry. B cell protein yield was optimised in a series of experiments using varying quantities of beads; the yield from 100ml peripheral blood was consistently sufficient to give measurable levels of pl,3g T activity. The data from this first series of assays does not show any statistically significant differences between patient and control pl,3g T activity when assessed by Student's unpaired t tests. The intra-assay CV for THP-1 pl,3g T activity was 32%, which is too high; ideally the CV should be less than 10%. The fact that there was a positive correlation between the pl,3gt activities of most test cell lysates and the internal control (THP-1) lysate is concerning and indicates a probable problem with the assay. Lectin binding studies performed on the serum IgA from both groups surprisingly showed no differences between patients and controls, which provides a possible explanation of the lack of difference in p 1,3GT activity. 4.5 Discussion The results failed to demonstrate a reduction in B cell pl,3gt activity in patients with IgAN when compared to controls, which was the initial hypothesis. There are two possible 88

107 explanations: (1) peripheral blood B cell pi,3gt activity in IgAN is normal and the results are indicative of this; (2 ) there is in fact a significant difference between patient and control peripheral blood B cell (31,3GT activity in IgAN but there is a problem with experimental design/methodology so that this difference was not apparent in this series of studies. I will discuss both possibilities but will start with some general points on the results, which may influence interpretation o f the data. The CV of THP-1 pl,3g T activity was poor at 32% and therefore cannot be said to be constant, which is a prerequisite of the assay system. There seemed to be a tendency for THP-1 activity to increase relative to U937 activity with time and certainly the last value was higher than previous values. If the CV is recalculated ignoring the last value it is greatly improved at 21%, suggesting that the final value at least is an outlier. The finding of poorly functioning control cell activity relative to the standard is also suggested by the correlations between test cell lysates and THP-1 lysate activity. Could the performance be improved? Because of the necessity to have plentiful THP-1 and U937 cells at all times during the period of experimentation and the serious consequences that would have ensued if cells were lost to infection or other events, cells were being cultured in three different incubators. During the series of experiments there was evidence to suggest (data not shown) that cells from different incubators were performing differently. For subsequent series the cell culture regime was tightened such that only cells from one incubator were used; these were inspected closely prior to centrifugation and changing of medium and, if healthy, were mixed, then resuspended. Medium was prepared in 125ml aliquots so it was not subjected to repeated warming, opening and possible contamination. Centrifugation and feeding occurred on set days, which had a fixed relationship to assay days to make constant the stage of the cell cycle at which the experiments took place. It will be seen in 89

108 Chapter Five that these changes led to improvements in intra-assay CV. The lack of difference in HA and W lectin binding between IgAl from patient and control groups was unexpected. The observed and well-described difference in lectin binding in IgAN is thought to be due to reduced terminal galactosylation on the hinge region. It is the investigation of this abnormality that generated the hypothesis of reduced activity of the responsible enzyme, P 1,3GT. From these lectin binding results it appears that the group of patients studied were not representative of IgAN patients overall. One would perhaps not expect to find abnormalities of pi,3g T activity in this sample o f IgAN patients even if demonstrated to occur in another population. To overcome this problem a larger sample was needed and indeed lectin studies were performed on stored serum of known IgAN patients. Those patients with known abnormal lectin binding profiles were specifically invited to take part in the subsequent bone marrow study. From the point of view of study design this is clearly not optimal but as the peripheral blood study was a prelude to the more important bone marrow study and all the patients studied had agreed to have BMs taken, it was not felt that they should be discarded. This was particularly undesirable, as it had not proved easy to recruit patients to a bone marrow study (perhaps unsurprisingly!). To make the subsequent study useful the sample needed to be augmented with the intention o f demonstrating abnormal serum lectin binding characteristics in the bone marrow study. There has already been considerable discussion about the benefits and disadvantages of the acceptor used in this study compared to that used in the study of Allen et al (1997). It is possible that aosm is a less effective acceptor for IgAl pl,3g T than degalactosylated IgAl hinge region fragments. The acceptor may have had a bearing on the functioning of the experimental system. A further potential problem was that it was not possible to 90

109 fractionate the cells sufficiently to study those of real interest, namely, IgAl plasma cells because of amount of cell lysate protein needed. pi,3g T is clearly not specific to these cells, being found also in T cells, monocytes and presumably all B cells. The previous study suggested that abnormalities of function are limited to B cells (Allen et al, 1997) and it is possible that a real difference in IgAl plasma cell pi,3g T activity was obscured by background activity. It is possible that a more specific acceptor (such as IgAl hinge region fragments) might have focused the assay onto the enzyme of interest, namely IgAl plasma cell pl,3g T by being a less effective acceptor for other pi,3gts. In addition to consideration of confounding problems alternative explanations of the results should be considered, namely that there is no difference between patient and control pl,3g T activity. Work from Japan suggested on the basis of ELISA studies using an anti- IgAl hinge antibody that the deficiency lies with N-acetylgalactosaminylation, ie the enzyme transferring the GalNAc moiety (Kokubo et al, 1999). Were this the case pi,3gt activity would be expected to be normal. This brings us back to the initial difficulties in establishing the nature o f glycosylation abnormalities in IgAN. Despite the negative results the assay had functioned largely as planned and the main purpose of this body of work was to study pi,3gt activity in the bone marrow of patients with IgAN. With the minor modifications described above and those described in the Methods section the next chapter details the similar series of experiments performed measuring pl,3g T activity o f bone marrow B cell precursors and appropriate controls. 91

110 Chapter Five pi,3gt activity in bone marrow and blood of patients and controls 5.1 Introduction The previous two chapters document the development and first application of an assay of pi,3g T activity using the incorporation of a radiolabelled substrate by enzyme in lysates derived from haematogenous cells. The application was largely successful but the results did not duplicate the results of the only previous attempt to measure (31,3GT activity in B cells in IgAN (Allen et al, 1997). Specific problems were addressed in the discussion of Chapter Four and as a result certain changes were made. The IgA deposited in the glomerular mesangium is polymeric IgAl (Lomax-Smith et al, 1983; Monteiro et al, 1985) and is now considered most likely to derive from the bone marrow (van den Wall Bake et al, 1988i; Harper et al, 1996; de Fijter et al, 1996) perhaps as a result of aberrant production of "mucosal" type antibody (Barratt et al, 1999), which occurs as a result of a putatively disordered marrow-mucosal axis (de Fijter et al, 1996; Allen et al, 1999iii). The abnormal glycosylation of circulating IgAl and specifically antibodies to "mucosal-type" antigens (eg to Hp) in IgAN may be related to its deposition in the mesangium (Allen et al, 2001). The hypothesis to be tested by this thesis was that abnormal hinge galactosylation may be explained by reduced B cell pi,3g T activity. The main purpose of this work therefore was to measure pl,3gt activity in the bone marrow of 92

111 patients with IgAN. Abnormalities of activity would be expected if this site were the origin of the nephritogenic IgAl and (31,3GT is the cause of aberrant glycosylation. This chapter details the series of experiments performed to measure pi,3g T activity in subsets of cells from the bone marrow of patients with IgAN and controls. pl,3g T activity was measured in bone marrow B cells and B cell precursors, which were captured by positive selection by antibody-bound magnetic beads, CD 19 as used in Chapter Four. Some preliminary studies were performed selecting for CD 138 positivity (a plasma cell marker) but this method yielded insufficient cells (see Table 5.2) and was therefore abandoned. The control cell lysates studied were reticulocytes and peripheral B cells from the same subjects. From studies on Tn syndrome reticulocytes were known to express pl,3g T (Hesford et al, 1981) and were positively selected using CD71 Dynabeads, which select for the transferrin receptor, expressed in high numbers on reticulocytes. (It is also found on proliferating leucocytes but is absent on resting leucocytes.) Peripheral blood B cell pl,3g T activity was restudied because of the methodological problems discussed in Chapter Four and because, in the event of bone marrow B cells expressing abnormal activity it would be important to know if this deficiency was limited to the bone marrow. The correlation between lectin binding and bone marrow and peripheral B cell pl,3gt activity was examined. It was hoped that by increasing the sample size the glycosylation profile would be representative of published abnormalities, that is increased binding of W and HA, signifying reduced terminal galactosylation o f hinge region glycans. 93

112 5.2 Subjects and Methods Subjects Twelve patients with biopsy proven IgAN under the care of the Department of Nephrology, Leicester General Hospital and 13 matched controls undergoing elective surgery were recruited for this study. IgAN patients were identified from the departmental database of patients with biopsyproven IgAN. Only those with a normal serum creatinine at last clinic review were approached. In order to keep the study group as homogenous as possible, non-caucasian patients and those with proteinuria greater than 2g/24h were excluded, as were patients with other systemic conditions such as diabetes and those with Henoch Schonlein purpura. A letter explaining the study and an invitation to participate was sent by post to the patients with a tear-off slip and a stamped self-addressed envelope. A follow-up telephone call was made to those who failed to reply. Initially 15 patients diagnosed in the previous three years were approached, of whom seven replied favourably and five ultimately had a bone marrow aspirate (not the same five as were venesected for the PB study). Further letters were sent to 16 patients diagnosed between three and five years earlier, of whom six replied positively and then underwent bone marrow aspirate. In addition three other patients who had previously been accommodating with departmental research were approached and agreed to the procedure. (31,3GT activity was measured on the first 12 aspirates. The IgAN group consisted of ten males and two females, with a median age of 39 years, range years. Because of disease progression between recruitment and performing the marrow study, one patient had a serum creatinine greater than pmol/l. 94

113 The controls were undergoing elective orthopaedic surgery under general anaesthetic in the Department o f Sports Medicine, Leicester General Hospital. They were fitter and younger than patients undergoing other forms of surgery and thus were well matched with the patient group. They were approached on the day prior to surgery and given an information leaflet about the research. Those that agreed were consented on the day of operation. Bone marrow aspirates were collected from seven males and six females with a median age of 37 years, range years, with no evidence of renal, immunological or systemic disease. Patient and control details are summarised in Table 5.1. The study was approved by the Leicestershire Ethics Committee and all subjects gave written informed consent to their participation Samples and controls Bone marrow For the patients with IgAN the bone marrow aspirates were collected under local anaesthesia with light intravenous sedation (2-10mg Midazolam depending on response). Samples were obtained from controls under general anaesthesia immediately before commencement of orthopaedic surgery (mostly surgery to ruptured Achilles tendons and ankle joints). Bone marrow was aspirated under aseptic conditions from the posterior iliac crest into syringes containing preservative-free heparin (100 U/ml bone marrow) and the samples were gently mixed. A relatively large amount of bone marrow, about 11ml, was needed for the study; immunohistochemistry was performed in order to demonstrate that the sample was bone marrow and not significantly diluted with peripheral blood. For the sake of clarity the bone marrow and peripheral blood protocols are shown in Appendix 2. 95

114 Patient Sex Age s.creat BP BP Rx ACE I Protein Haemat- No. of drugs or ARB uria uria I M * * * * * U F /70 0 No 0 3+ III M /75 2 No 0 0 IV M /100 1 No 1+ 0 V M /95 4 Yes VI M /80 0 No 0 0 VII M /100 1 No VIII F /84 0 No IX M /90 0 No 2+ 0 X M /78 0 No XI M /100 1 No XII M /80 1 Yes Control Sex Age s.creat BP BP Rx ACE I Protein Haemat- No. of drugs or ARB uria uria 1 M /75 0 No 2 F /70 0 No 3 F /70 0 No 4 M /60 0 No 5 F /72 0 No 6 M 39 * 110/60 0 No 7 F /60 0 No 8 M 52 * 115/70 0 No 9 F 35 * 125/80 0 No 10 M 17 * 115/60 * * 11 M 37 * 123/85 0 No 12 F 26 * 95/55 0 No 13 M 75 * 145/60 0 No Table 5.1 Individual details of patients and controls undergoing bone marrow aspirate. * Data unavailable 96

115 10ml of HBSS was added to the aspirate and was vortexed hard to disrupt clumps of cells. Initial centrifugation took place (500g, 5 min, RT) then the cells were resuspended in HBSS and 1ml removed for separation of reticulocytes, which was centrifuged and transferred into THP-1 culture medium (Appendix 1). The remaining bone marrow was separated by density gradient centrifugation (Chapter 2.2) the cells washed, resuspended in THP-1 medium and kept under culture conditions overnight. Peripheral blood 60ml venous blood was taken into syringes containing preservative-free heparin (10 U/ml), was separated by density gradient centrifugation (Chapter 2.1) and kept under culture conditions overnight. The next day the bone marrow mononuclear cells (BMMC) were separated into bone marrow B cells and bone marrow reticulocytes using positive selection with CD 19 and CD71 (respectively) magnetic beads (Dynabeads, Dynal Ltd, Wirral, UK) as detailed in Chapter Two, (2.5). Peripheral blood B cells were selected using CD 19 Dynabeads. The volumes of beads used are discussed in section After selection all cells were lysed with TBS/Triton X 100 as described (Chapter 2.5.5) using the smallest volume that would yield the required volume of lysate. The cells for "standard" (U937) and "standard control" (THP-1) lysates were prepared as described in Chapter Two (2.6). Protein content of all lysate samples was measured as described in Chapter 2.9. The lysates were then ready for P 1,3GT measurement. 97

116 5.2.3 Immunohistochemistry Bone marrow smears were made from fresh marrow (Chapter 2.8) and cytospins were made of BMMCs immediately after density gradient centrifugation and of the cells selected by CD 19 beads after overnight incubation (Chapter 2.7). The smears and cytospins were stained using monoclonal primary antibodies to cell surface markers and alkaline phosphatase-labelled anti-mouse secondary antibodies (Chapter 2.7.2). Colour was developed with a chromogenic substrate. The antibodies used were anti-cd3, anti-cd14, anti-cd 138 and anti-dr (against T cells, monocytes, plasma cells and HLA Class II respectively). Further slides were stained with MGG (Chapter 2.7.1) for morphometry. Cells were counted using light microscopy with the aid of a cell counter Determination o f optimal volume o f Dynabeads As for pi,3g T assay on PBMCs described in Chapter Four, it was necessary to optimise the B cell lysate protein yield. To this end an evaluation was performed using different volumes of CD 19 Dynabeads (to assess the maximum B cell protein yield). BMMCs were prepared from subjects A and B, from 22ml and 10ml of bone marrow aspirate respectively on separate occasions. BMMCs were divided into 4 equal aliquots (Subject A) and 2 aliquots (Subject B). The first study (Subject A) was performed using Dynabead volumes of 50pl, 75pl, loopl, and 125pl per 5.5ml bone marrow. B cell separation with the beads took place as previously described (Chapter 2.6). The cells were lysed with 37.5pl TBS/Triton X 100, the beads were immobilised and the cell lysate was aspirated. Protein concentration was measured as previously described (Chapter 2.9). The maximum protein yield was achieved using 125pl beads so the second study (Subject B) was undertaken using 125pi and 160pl CD 19 beads per 5ml aspirate using the same techniques. As a result 98

117 o f this assay 150pl CD 19 beads were used per 5ml of bone marrow aspirate, which equated to 300pl for 10ml aspirate. For PBMC B cells equivalent volumes of CD 19 beads were used as in Chapter However, because the assays were performed on half volumes, only 60ml blood was taken and 150pl of beads were used. The volumes for CD71 beads used for separation of reticulocytes were in accordance with manufacturer's instructions. An initial study took place using 50pl beads for 2ml aspirate as described in Chapter 2.6 using PBS/0.1% BSA as wash buffer as recommended, which yielded 170pg o f reticulocyte lysate protein. A trial pl,3g T assay was undertaken on reticulocyte lysate obtained from using 75pi beads/ml aspirate and generated (31,3GT activity on the straight part of the standard curve and was therefore the volume used for all subsequent assays pl,3g T activity o f bone marrow B cell precursors and controls The assays (as detailed in Chapter 3.2.4) were carried out on 15 occasions over a period of three months; on each occasion samples from between one and three subjects were used, for logistical reasons it was not always possible to run patient and control samples simultaneously. The dates and order of assays are shown in Table 5.5. For each subject assays were performed on one concentration of each test lysate. A U937 standard curve was prepared and lysates of three concentrations of THP-1 were used as internal controls. All control cells were cultured in one incubator for the entire period. 99

118 5.2.6 Reproducibility o f p l,3 G T activity The validity of comparisons made between assays run on different days was dependent on the constancy of the relationship between the activities of the internal standard (THP-1 cell lysate) and the standard lysate (U937). The inter-assay CV was therefore calculated for the THP-1 lysate activity in each assay over the series of 15 assays Lectin binding o f serum o f IgA 1 The binding of HA and W lectins to each subject s serum was assessed as described in Chapter All samples were run in a single assay on a single immunoplate to allow comparison. The correlation between lectin binding and bone marrow and peripheral B cell pl,3g T activity was examined Statistical analysis pl,3g T activities of all three cell lysates in IgAN and controls were compared by unpaired Student's t tests, as were lectin binding results of serum IgA. Linear regression analysis was carried out to probe the functioning of the control lysate activity by looking at the relationship between pi,3g T activity of test and THP-1 lysates. Linear regression analysis was also used to investigate the relationships between pl,3g T activity o f each cell type and lectin binding of serum IgA from the same subject in IgAN and controls. Results quoted in the text are mean values of IgAN or control values. 100

119 5.3 Results Immunohistochemistry Cell counting prior to any processing revealed 3.85 x 109 cells/ml bone marrow aspirate. After density gradient centrifugation there were 6.5 x 106 mononuclear cells/ml aspirate, representing a reduction in cell number by a factor of 600. The proportions of cells staining positive for the cell surface markers at the different stages of processing are shown in Table 5.2. CD3+ve CD14+Ve CD138+Ve DR+ve Smears 11% 7% < 1% 2 0 % BMMCs 34% 29% 36% Post CD 19 beads negative negative negative 7/10 Table 5.2 The percentage of bone marrow cells staining positive by immunohistochemistry at three different stages of separation. CD3 is expressed by T cells, CD 14 by monocytes, CD 138 by plasma cells and DR by HLA Class II cells including B cells and monocytes. The percentages are means for multiple cell counts. There were few cells present after treatment with Dynabeads and the absolute counts are therefore presented. Many nucleated red cells were present on the smears. The other cells present after processing were nucleated cells, presumably precursors not mature enough to express these markers. Initially erythrocytes compromised the majority cell type, which explains why only about 30% of cells were positive for the surface markers used (DR+ cells include monocytes, which are also CD14+). Disappointingly, there were very few plasma cells present, precluding the sole use of these cells for measurement of pi,3gt activity. The proportions o f cells expressing T cell markers were significantly less than for PBMCs, as would be 101

120 expected for bone marrow. For example, while 65% of PBMCs were T cells, only 34% of BMMCs expressed T cell markers (see Table 4.1 to compare bone marrow and peripheral blood cell types). A large number of cells prior to Dynabead separation were likely to be immature precursor cells which did not stain positive for any of the markers used, hence the smaller proportion of cells that stained compared to PBMCs in Chapter Four. After Dynabead treatment there were relatively few cells remaining; 7/10 of the cells seen were positive for DR but negative for CD 14 and were morphologically lymphocytes, the expected staining characteristics o f B cells and B cell precursors. MGG staining of cytospins of bone marrow cells selected with CD71 Dynabeads revealed nucleated cells bound to beads that were morphologically reticulocytes. There was much red cell contamination, which precluded cell counting and photography Determination o f optimal volume o f Dynabeads The results from the experiments to maximise the yield of bone marrow B cell lysate protein are shown in Table 5.3. In the first assay the protein yield increased with increasing volumes of beads and had not reached a plateau at the top concentration used. Using both 125 and 160pl CD 19 Dynabeads/5ml aspirate yielded >100pg lysate protein/10ml aspirate, which had been sufficient to put pl,3g T activity onto the straight part of the standard curve when half volumes o f substrate and lysate were used, as described in Chapter Because the protein yield increased within the range used, 150pl beads/5ml aspirate were ultimately used for bone marrow B cell 31,3GT measurement, ie. 300pl for 10ml aspirate. 102

121 S ubject Bead volum e (Hi) B cell protein (pg/m l) Lysis volume (Hi) B cell protein yield/aliquot (Hg) Total achievable protein (Hg) A B Tables 5.3 M axim um B cell protein yield using a range of volumes of CD19 m agnetic beads. For Subject A 5.5ml bone marrow aliquots were used, for Subject B each sample was 5ml. The total achievable protein is the calculated yield if the corresponding volume of beads was used on 10ml of bone marrow pl,3g T activity o f bone marrow B cell precursors Table 5.4 shows pi,3gt activity for patient and control bone marrow and peripheral blood B cell lysates and that of corresponding reticulocytes. No statistically significant differences were found between patient and control lysate activity using Student's unpaired t test, shown in Table 5.4. There was a positive correlation between patient blood and BM p!3gt activity (p<0.05) but not for controls. The relevance of this relationship is unclear. 103

122 P atient/ Reticulocyte Peripheral B cell Bone m arrow B cell C ontrol pl,3 G T activity pl,3g T activity p l,3 G T activity num ber: (AU/pg) (AU/pg) (AU/pg) Patient C ontrol Patient Control P atient C ontrol * M ean SD sem P Table 5.4 pi,3g T activity of patient and control bone m arrow -B cell, peripheral blood-b cell and reticulocyte lysates in AU/pg. No statistically significant differences were found by Student's unpaired t tests comparing patient and control values. The correlation coefficients (r) relating the levels of pl3gt in blood and bone marrow samples for patients and controls were (p<0.05, n=12) and (NS, n=13) respectively. * Reticulocyte pi,3g T activity was not measured on the first control bone marrow. 104

123 5.3.4 Reproducibility o f pl,3g T activity The data for THP-1 pl,3gt activity through the 3 month series of experiments are shown in Table 5.5. The CV was 16%. Experim ent date Subjects Control Control Control Control Patient I Control Patient II Patient III 0.72 Control Patient IV 0.80 Patient V Patient VI 0.95 Control Control Control Patient VII 0.80 Control 10 Control Patient VIII Patient EX 0.93 Control Patient X 0.86 Patient XI Patient XII Control THP-1 activity (AU/pg) M ean 0.86 SD 0.14 CV 16% Table 5.5 T able show ing th e d ates and sequence of experim ents and the in te r assay coefficient of variation (CV) of THP-1 p i,3g T activity over the three month series of experiments. 105

124 5.3.5 Relationship between the fil,3g T activity o f test cell- and THP-1 lysates The correlation coefficients and p values from linear regression analysis performed to examine the relationship between (31,3GT activity of patient and control cell lysates and that of THP-1 lysate are shown in Table 5.6. Unlike the previous set of experiments on peripheral blood (Chapter Four) there were no correlations between (31,3GT activity of any test cell lysates and the control cell lysate, THP-1. pi,3g T activity r value p value Patient Reticulocyte vs. THP PB B cell vs. THP BM B cell vs. THP Control Reticulocyte vs. THP PB B cell vs. THP BM B cell vs. THP Table 5.6 The relationships between test cell 01,3GT activity and that of THP-1 lysates. There were no significant associations between any test lysate activity and THP-1 lysate activity. 106

125 5.3.6 Lectin b inding o f serum of IgA 1 The binding of HA and W lectins are demonstrated in Table 5.7. Unfortunately, for logistic reasons, serum was only available for 11 patients and 11 controls. Patient W and HA binding was significantly higher than that of controls. H A lectin binding (OD) W lectin binding (OD) P atient Control Patient Control mean ' sem P 0.014* 0.003* T able 5.7 L ectin binding of p atien t and control IgA (expressed in O D 492). Student s unpaired t tests indicate that binding of W and HA are significantly higher in the IgAN group, indicating increased exposure of GalNAc. VV - Vicia villosa (GalNAc) HA - Helix aspersa (GalNAC) * - significant difference 107

126 5.3.7 Correlation between J31,3GT activity and lectin binding The correlation coefficients and p values from linear regression analysis performed to examine the relationship between (31,3GT activity of patient and control cell lysates and HA and W lectin binding are shown in Table 5.8. IgAN PB B cell P 1,3GT activity vs w PB B cell pl,3g T activity vs HA BM B cell pi,3g T activity vs W B M B cell pi,3gt activity vs HA n m o* V. U X ^ C ontrol PB B cell pl,3gt activity vs W PB B cell pl,3g T activity vs HA * BM B cell pl,3g T activity vs W BM B cell pi,3g T activity vs HA * r P Table 5.8 The relationships between test cell pl,3g T activities and HA and W lectin binding. There were significant correlations between patient bone marrow B cell-, control bone marrow B cell- and peripheral B cell-pi,3gt activities and HA binding. Interestingly, the correlations are negative for controls but positive for IgAN patients. The results are demonstrated graphically for the highlighted cells. * denotes a significant correlation The correlations between bone marrow B cell pi,3gt activity and HA lectin binding for patients and controls are shown graphically in Figure

127 Significant correlations were found between HA binding and patient and control bone marrow B cell (31,3GT activity and control peripheral B cell enzyme activity. In the controls higher enzyme activity was associated with reduced GalNAc exposure and presumed increased terminal galactosylation, as might be expected whereas an unexpectedly positive correlation was found for patient BM enzyme activity and HA binding, indicating increased GalNAc exposure, ie. decreased exposure of terminal galactose in subjects with higher (31,3GT activity. 2.4 co IgAN r = p = Control r = p = (31,3GT activity of BM B cells (AU/jug protein Figure 5.1 The relationship between bone m arrow B cell pl,3g T activity in IgAN patients and controls an d HA lectin binding. 109